DNA Polymerase α (swi7) and the Flap Endonuclease Fen1 (rad2) Act Together in the S-Phase Alkylation Damage Response in S. pombe

Polymerase α is an essential enzyme mainly mediating Okazaki fragment synthesis during lagging strand replication. A specific point mutation in Schizosaccharomyces pombe polymerase α named swi7-1, abolishes imprinting required for mating-type switching. Here we investigate whether this mutation confers any genome-wide defects. We show that the swi7-1 mutation renders cells hypersensitive to the DNA damaging agents methyl methansulfonate (MMS), hydroxyurea (HU) and UV and incapacitates activation of the intra-S checkpoint in response to DNA damage. In addition we show that, in the swi7-1 background, cells are characterized by an elevated level of repair foci and recombination, indicative of increased genetic instability. Furthermore, we detect novel Swi1-, -Swi3- and Pol α- dependent alkylation damage repair intermediates with mobility on 2D-gel that suggests presence of single-stranded regions. Genetic interaction studies showed that the flap endonuclease Fen1 works in the same pathway as Pol α in terms of alkylation damage response. Fen1 was also required for formation of alkylation- damage specific repair intermediates. We propose a model to explain how Pol α, Swi1, Swi3 and Fen1 might act together to detect and repair alkylation damage during S-phase.     

Role of STN1 and DNA Polymerase α in Telomere Stability and Genome-Wide Replication in Arabidopsis

The CST (Cdc13/CTC1-STN1-TEN1) complex was proposed to have evolved kingdom specific roles in telomere capping and replication. To shed light on its evolutionary conserved function, we examined the effect of STN1 dysfunction on telomere structure in plants. STN1 inactivation in Arabidopsis leads to a progressive loss of telomeric DNA and the onset of telomeric defects depends on the initial telomere size. While EXO1 aggravates defects associated with STN1 dysfunction, it does not contribute to the formation of long G-overhangs. Instead, these G-overhangs arise, at least partially, from telomerase-mediated telomere extension indicating a deficiency in C-strand fill-in synthesis. Analysis of hypomorphic DNA polymerase α mutants revealed that the impaired function of a general replication factor mimics the telomeric defects associated with CST dysfunction. Furthermore, we show that STN1-deficiency hinders re-replication of heterochromatic regions to a similar extent as polymerase α mutations. This comparative analysis of stn1 and pol α mutants suggests that STN1 plays a genome-wide role in DNA replication and that chromosome-end deprotection in stn1 mutants may represent a manifestation of aberrant replication through telomeres.     

Paraspeckle Protein 1 (PSPC1) Is Involved in the Cisplatin Induced DNA Damage Response—Role in G1/S Checkpoint

Paraspeckle protein 1 (PSPC1) was first identified as a structural protein of the subnuclear structure termed paraspeckle. However, the exact physiological functions of PSPC1 are still largely unknown. Previously, using a proteomic approach, we have shown that exposure to cisplatin can induce PSPC1 expression in HeLa cells, indicating the possible involvement for PSPC1 in the DNA damage response (DDR). In the current study, the role of PSPC1 in DDR was examined. First, it was found that cisplatin treatment could indeed induce the expression of PSPC1 protein. Abolishing PSPC1 expression by siRNA significantly inhibited cell growth, caused spontaneous cell death, and increased DNA damage. However, PSPC1 did not co-localize with γH2AX, 53BP1, or Rad51, indicating no direct involvement in DNA repair pathways mediated by these molecules. Interestingly, knockdown of PSPC1 disrupted the normal cell cycle distribution, with more cells entering the G2/M phase. Furthermore, while cisplatin induced G1/S arrest in HeLa cells, knockdown of PSPC1 caused cells to escape the G1/S checkpoint and enter mitosis, and resulted in more cell death. Taken together, these observations indicate a new role for PSPC1 in maintaining genome integrity during the DDR, particularly in the G1/S checkpoint.     

Protein Arginine Methyltransferase 7 Regulates Cellular Response to DNA Damage by Methylating Promoter Histones H2A and H4 of the Polymerase δ Catalytic Subunit Gene, POLD1

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage.     

Identification of RNF8 as a Ubiquitin Ligase Involved in Targeting the p12 Subunit of DNA Polymerase δ for Degradation in Response to DNA Damage

DNA polymerase δ consists of four subunits, one of which, p12, is degraded in response to DNA damage through the ubiquitin-proteasome pathway. However, the identities of the ubiquitin ligase(s) that are responsible for the proximal biochemical events in triggering proteasomal degradation of p12 are unknown. We employed a classical approach to identifying a ubiquitin ligase that is involved in p12 degradation. Using UbcH5c as ubiquitin-conjugating enzyme, a ubiquitin ligase activity that polyubiquitinates p12 was purified from HeLa cells. Proteomic analysis revealed that RNF8, a RING finger ubiquitin ligase that plays an important role in the DNA damage response, was the only ubiquitin ligase present in the purified preparation. In vivo, DNA damage-induced p12 degradation was significantly reduced by shRNA knockdown of RNF8 in cultured human cells and in RNF8^−/− mouse epithelial cells. These studies provide the first identification of a ubiquitin ligase activity that is involved in the DNA damage-induced destruction of p12. The identification of RNF8 allows new insights into the integration of the control of p12 degradation by different DNA damage signaling pathways.     

A Role for DNA Polymerase μ in the Emerging DJH Rearrangements of the Postgastrulation Mouse Embryo

The molecular complexes involved in the nonhomologous end-joining process that resolves recombination-activating gene (RAG)-induced double-strand breaks and results in V(D)J gene rearrangements vary during mammalian ontogeny. In the mouse, the first immunoglobulin gene rearrangements emerge during midgestation periods, but their repertoires have not been analyzed in detail. We decided to study the postgastrulation DJ_H joints and compare them with those present in later life. The embryo DJ_H joints differed from those observed in perinatal life by the presence of short stretches of nontemplated (N) nucleotides. Whereas most adult N nucleotides are introduced by terminal deoxynucleotidyl transferase (TdT), the embryo N nucleotides were due to the activity of the homologous DNA polymerase μ (Polμ), which was widely expressed in the early ontogeny, as shown by analysis of Polμ^−/− embryos. Based on its DNA-dependent polymerization ability, which TdT lacks, Polμ also filled in small sequence gaps at the coding ends and contributed to the ligation of highly processed ends, frequently found in the embryo, by pairing to internal microhomology sites. These findings show that Polμ participates in the repair of early-embryo, RAG-induced double-strand breaks and subsequently may contribute to preserve the genomic stability and cellular homeostasis of lymphohematopoietic precursors during development.The adaptive immune system is characterized by the great diversity of its antigen receptors, which result from the activities of enzymatic complexes that cut and paste the genomic DNA of antigen receptor loci. The nonhomologous end-joining (NHEJ) machinery is then recruited to repair the double-strand DNA breaks (DSBs) inflicted by the products of the recombination-activating genes (RAGs) (45, 65). Within B cells, each immunoglobulin (Ig) receptor represents a singular shuffling of two heavy (H) and two light (L) chains, which are derived from the recombination of V, D, and J gene segments of the IgH locus and of V and J for IgL (71). Besides these combinatorial possibilities, most Ig variability derives from extensive processing of the coding ends, including exonucleolytic trimming of DNA ends, together with the addition of palindromic (P) nucleotides templated by the adjacent germ line sequence and of nontemplated (N) nucleotides secondary to the activity of the terminal deoxynucleotidyl transferase (TdT), a lymphoid-specific member of family X of DNA polymerases (reviewed in reference 56). During B-lineage differentiation, IgH rearrangements occur before those of the IgL locus, and D-to-J_H rearrangements precede V-to-DJ_H rearrangements (62). DJ_H joints are formed in any of the three open reading frames (ORFs). ORF1 is predominantly used in mature Igs, ORF2 is transcribed as a Dμ protein that provides negative signals to the B-cell precursors, and ORF3 frequently leads to stop codons (32, 33, 37). Germ line V, D, and J gene segments display short stretches of mutually homologous nucleotides (SSH), which are frequently used in gene rearrangements during perinatal periods, when N additions are absent (27, 32, 55, 57). The actual Ig V-region repertoires represent both the results of the NHEJ process associated with genomic VDJ recombination and those of antigen-independent and -dependent selection events. Although the core NHEJ components (Ku-Artemis-DNA-PK and XLF-XRCC4-DNA ligase IV) are by themselves able to join RAG-induced, incompatible DNA ends, family X DNA polymerases can be recruited to fill gaps created by imprecise coding ends with 3′ overhangs (DNA polymerase μ [Polμ] and Polλ) and/or to promote diversity through the addition of N nucleotides (TdT) (34, 56).The lymphoid differentiation pathways and clonotypic repertoires are developmentally regulated and differ between the embryo-fetal and adult periods (2, 44, 68). The perinatal B cells result from a wave of B lymphopoiesis occurring during the last third of mouse gestation (13, 14, 21, 70). Perinatal V_H gene usage differs from that predominating in the adult (1, 69), and the former VDJ joints rarely display N additions, leading to V-region repertoires enriched in multi- and self-reactive specificities (36, 40). The program of B-cell differentiation starts at embryonic days 10 to 11 (E10 to E11) in embryo hematopoietic sites, after the emergence of multipotent progenitors (at E8.5 to E9.5) (18, 19, 23, 31, 51, 73). DJ_H rearrangements were detected in these early embryos, whereas full VDJ_H sequences were not observed before E14 (14, 18, 51, 66), when VJκ rearrangements were also found (63). The earliest mouse DJ_H/VDJ_H Ig sequences analyzed to date corresponded to late fetuses (E16) (14, 53). We reasoned that the true baseline of the Ig rearrangement process occurs in midgestation embryos, when the first DJ_Hs are not yet transcribed and, consequently, not subjected to selection and are conditioned only for the evolutionarily established and developmentally regulated usage of distinct NHEJ machineries.We report here the sequence profiles of the earliest embryo E10 to E12 DJ_H joints. Unexpected frequencies of embryonic DJ_H joints bearing N nucleotides, in the absence of detectable TdT expression, were found. Moreover, the embryo DJ_H joints lacking N nucleotides (N⁻) used fewer SSH to recombine than newborn DJ_Hs, and these SSH were widely dispersed along the embryo D sequences, in contrast to the most joint-proximal ones, which predominated in newborn DJ_Hs. Considering that Polμ is the closest relative of TdT (42% amino acid identity) (22), which is able to introduce N nucleotides in vitro (4, 22, 34, 39, 49) and to join DNA ends with minimal or even null complementarity (17, 58), and that it is expressed in early-embryo organs, we decided to investigate its putative contribution to the first embryo DJ_H joints. The DJ_H joints obtained from Polμ^−/− embryos (48) showed a significant reduction of N nucleotides compared to wild-type (WT) embryos. Moreover, highly preserved DJ_H joints (with <3 deleted nucleotides) were selectively depleted in the Polμ^−/− mouse embryos, while the remaining DJ_Hs preferentially relied upon longer stretches of homology for end ligation. These findings support the idea that Polμ is active during early-embryo DJ_H rearrangements and that both its template-dependent and -independent ambivalent functions may be used to fill in small nucleotide gaps generated after asymmetric hairpin nicking and also to extend coding ends via a limited TdT-like activity.     

Involvement of the Yeast DNA Polymerase δ in DNA Repair in Vivo

The POL3 encoded catalytic subunit of DNA polymerase δ possesses a highly conserved C-terminal cysteine-rich domain in Saccharomyces cerevisiae. Mutations in some of its cysteine codons display a lethal phenotype, which demonstrates an essential function of this domain. The thermosensitive mutant pol3-13, in which a serine replaces a cysteine of this domain, exhibits a range of defects in DNA repair, such as hypersensitivity to different DNA-damaging agents and deficiency for induced mutagenesis and for recombination. These phenotypes are observed at 24°, a temperature at which DNA replication is almost normal; this differentiates the functions of POL3 in DNA repair and DNA replication. Since spontaneous mutagenesis and spontaneous recombination are efficient in pol3-13, we propose that POL3 plays an important role in DNA repair after irradiation, particularly in the error-prone and recombinational pathways. Extragenic suppressors of pol3-13 are allelic to sdp5-1, previously identified as an extragenic suppressor of pol3-11. SDP5, which is identical to HYS2, encodes a protein homologous to the p50 subunit of bovine and human DNA polymerase δ. SDP5 is most probably the p55 subunit of Polδ of S. cerevisiae and seems to be associated with the catalytic subunit for both DNA replication and DNA repair.     

Purification of the α Subunit of Avian Myeloblastosis Virus DNA Polymerase by Polyuridylic Acid-Sepharose

The alpha subunit of the avian myeloblastosis virus DNA polymerase could be readily purified to near homogeneity using a polyuridylic acid-Sepharose column chromatography step.     

The Role of Dbf4-Dependent Protein Kinase in DNA Polymerase ζ-Dependent Mutagenesis in Saccharomyces cerevisiae

The yeast Dbf4-dependent kinase (DDK) (composed of Dbf4 and Cdc7 subunits) is an essential, conserved Ser/Thr protein kinase that regulates multiple processes in the cell, including DNA replication, recombination and induced mutagenesis. Only DDK substrates important for replication and recombination have been identified. Consequently, the mechanism by which DDK regulates mutagenesis is unknown. The yeast mcm5-bob1 mutation that bypasses DDK’s essential role in DNA replication was used here to examine whether loss of DDK affects spontaneous as well as induced mutagenesis. Using the sensitive lys2ΔA746 frameshift reversion assay, we show DDK is required to generate “complex” spontaneous mutations, which are a hallmark of the Polζ translesion synthesis DNA polymerase. DDK co-immunoprecipitated with the Rev7 regulatory, but not with the Rev3 polymerase subunit of Polζ. Conversely, Rev7 bound mainly to the Cdc7 kinase subunit and not to Dbf4. The Rev7 subunit of Polζ may be regulated by DDK phosphorylation as immunoprecipitates of yeast Cdc7 and also recombinant Xenopus DDK phosphorylated GST-Rev7 in vitro. In addition to promoting Polζ-dependent mutagenesis, DDK was also important for generating Polζ-independent large deletions that revert the lys2ΔA746 allele. The decrease in large deletions observed in the absence of DDK likely results from an increase in the rate of replication fork restart after an encounter with spontaneous DNA damage. Finally, nonepistatic, additive/synergistic UV sensitivity was observed in cdc7Δ pol32Δ and cdc7Δ pol30-K127R,K164R double mutants, suggesting that DDK may regulate Rev7 protein during postreplication “gap filling” rather than during “polymerase switching” by ubiquitinated and sumoylated modified Pol30 (PCNA) and Pol32.     

An Iron–Sulfur Cluster in the Polymerase Domain of Yeast DNA Polymerase ε

DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Polε from Saccharomyces cerevisiae is composed of four subunits—Pol2, Dpb2, Dpb3, and Dpb4. Here, we report the presence of a [Fe-S] cluster directly within the active polymerase domain of Pol2 (residues 1–1187). We show that binding of the [Fe-S] cluster is mediated by cysteines in an insertion (Pol2^ins) that is conserved in Pol2 orthologs but is absent in the polymerase domains of Polα, Polδ, and Polζ. We also show that the [Fe-S] cluster is required for Pol2 polymerase activity but not for its exonuclease activity. Collectively, our work suggests that Polε is perhaps more sensitive than other DNA polymerases to changes in oxidative stress in eukaryotic cells.     

Role of the Polymerase ? sub-unit DPB2 in DNA replication,cell cycle regulation and DNA damage response in Arabidopsis

Faithful DNA replication maintains genome stability in dividing cells and from one generation to the next. This is particularly important in plants because the whole plant body and reproductive cells originate from meristematic cells that retain their proliferative capacity throughout the life cycle of the organism. DNA replication involves large sets of proteins whose activity is strictly regulated, and is tightly linked to the DNA damage response to detect and respond to replication errors or defects. Central to this interconnection is the replicative polymerase DNA Polymerase ϵ (Pol ϵ) which participates in DNA replication per se, as well as replication stress response in animals and in yeast. Surprisingly, its function has to date been little explored in plants, and notably its relationship with DNA Damage Response (DDR) has not been investigated. Here, we have studied the role of the largest regulatory sub-unit of Arabidopsis DNA Pol ϵ: DPB2, using an over-expression strategy. We demonstrate that excess accumulation of the protein impairs DNA replication and causes endogenous DNA stress. Furthermore, we show that Pol ϵ dysfunction has contrasting outcomes in vegetative and reproductive cells and leads to the activation of distinct DDR pathways in the two cell types.     

Antimutator Mutations in the α Subunit of Escherichia Coli DNA Polymerase III: Identification of the Responsible Mutations and Alignment with Other DNA Polymerases

The dnaE gene of Escherichia coli encodes the DNA polymerase (α subunit) of the main replicative enzyme, DNA polymerase III holoenzyme. We have previously identified this gene as the site of a series of seven antimutator mutations that specifically decrease the level of DNA replication errors. Here we report the nucleotide sequence changes in each of the different antimutator dnaE alleles. For each a single, but different, amino acid substitution was found among the 1,160 amino acids of the protein. The observed substitutions are generally nonconservative. All affected residues are located in the central one-third of the protein. Some insight into the function of the regions of polymerase III containing the affected residues was obtained by amino acid alignment with other DNA polymerases. We followed the principles developed in 1990 by M. Delarue et al. who have identified in DNA polymerases from a large number of prokaryotic and eukaryotic sources three highly conserved sequence motifs, which are suggested to contain components of the polymerase active site. We succeeded in finding these three conserved motifs in polymerase III as well. However, none of the amino acid substitutions responsible for the antimutator phenotype occurred at these sites. This and other observations suggest that the effect of these mutations may be exerted indirectly through effects on polymerase conformation and/or DNA/polymerase interactions.     

Cloning and Characterization of the Human Mitochondrial DNA Polymerase, DNA Polymerase γ

The nuclear-encoded DNA polymerase γ (DNA POLγ) is the sole DNA polymerase required for the replication of the mitochondrial DNA. We have cloned the cDNA for human DNA POLγ and have mapped the gene to the chromosomal location 15q24. Additionally, the DNA POLγ gene fromDrosophila melanogasterand a partial cDNA for DNA POLγ fromGallus gallushave been cloned. The predicted human DNA POLγ polypeptide is 1239 amino acids, with a calculated molecular mass of 139.5 kDa. The human amino acid sequence is 41.6, 43.0, 48.7, and 77.6% identical to those ofSchizosaccharomyces pombe, Saccharomyces cerevisiae, Drosophila melanogaster,and the C-terminal half ofG. gallus,respectively. Polyclonal antibodies raised against the polymerase portion of the protein reacted specifically with a 140-kDa protein in mitochondrial extracts and immunoprecipitated a protein with DNA POLγ like activity from mitochondrial extracts. The human DNA POLγ is unique in that the first exon of the gene contains a CAG₁₀trinucleotide repeat.     

The C-terminal Domain of the DNA Polymerase Catalytic Subunit Regulates the Primase and Polymerase Activities of the Human DNA Polymerase α-Primase Complex

The initiation of DNA synthesis during replication of the human genome is accomplished primarily by the DNA polymerase α-primase complex, which makes the RNA-DNA primers accessible to processive DNA pols. The structural information needed to understand the mechanism of regulation of this complex biochemical reaction is incomplete. The presence of two enzymes in one complex poses the question of how these two enzymes cooperate during priming of DNA synthesis. Yeast two-hybrid and direct pulldown assays revealed that the N-terminal domain of the large subunit of primase (p58N) directly interacts with the C-terminal domain of the catalytic subunit of polα (p180C). We found that a complex of the C-terminal domain of the catalytic subunit of polα with the second subunit (p180C-p70) stimulated primase activity, whereas the whole catalytically active heterodimer of polα (p180ΔN-p70) inhibited RNA synthesis by primase. Conversely, the polα catalytic domain without the C-terminal part (p180ΔN-core) possessed a much higher propensity to extend the RNA primer than the two-subunit polα (p180ΔN-p70), suggesting that p180C and/or p70 are involved in the negative regulation of DNA pol activity. We conclude that the interaction between p180C, p70, and p58 regulates the proper primase and polymerase function. The composition of the template DNA is another important factor determining the activity of the complex. We have found that polα activity strongly depends on the sequence of the template and that homopyrimidine runs create a strong barrier for DNA synthesis by polα.     

Role of the Y-Family DNA Polymerases YqjH and YqjW in Protecting Sporulating Bacillus subtilis Cells from DNA Damage

The role played by the Y-family DNA polymerases YqjH and YqjW in protecting sporulating cells of Bacillus subtilis from DNA damage was determined. The absence of either yqjH and/or yqjW not only reduced sporulation efficiency but also sensitized the sporulating cells to hydrogen peroxide, tert-butylhydroperoxide (t-BHP), mitomycin-C (M-C), and UV-C radiation. Moreover, these DNA-damaging agents increased the mutation frequency of wild-type sporulating cells to 4-azaleucine, but the production of mutants was YqjH- and YqjW-dependent. In conclusion, the results presented here indicate that YqjH/YqjW-dependent-translesion synthesis (TLS) operates in sporulating B. subtilis cells and contributes in processing spontaneous and artificially induced genetic damage, which is apparently required for an efficient sporulation process.     

The Translesion Polymerase ζ Has Roles Dependent on and Independent of the Nuclease MUS81 and the Helicase RECQ4A in DNA Damage Repair in Arabidopsis

DNA polymerase zeta catalytic subunit REV3 is known to play an important role in the repair of DNA damage induced by cross-linking and methylating agents. Here, we demonstrate that in Arabidopsis (Arabidopsis thaliana), the basic polymerase activity of REV3 is essential for resistance protection against these different types of damaging agents. Interestingly, its processivity is mainly required for resistance to interstrand and intrastrand cross-linking agents, but not alkylating agents. To better define the role of REV3 in relation to other key factors involved in DNA repair, we perform epistasis analysis and show that REV3-mediated resistance to DNA-damaging agents is independent of the replication damage checkpoint kinase ataxia telangiectasia-mutated and rad3-related homolog. REV3 cooperates with the endonuclease MMS and UV-sensitive protein81 in response to interstrand cross links and alkylated bases, whereas it acts independently of the ATP-dependent DNA helicase RECQ4A. Taken together, our data show that four DNA intrastrand cross-link subpathways exist in Arabidopsis, defined by ATP-dependent DNA Helicase RECQ4A, MMS and UV-sensitive protein81, REV3, and the ATPase Radiation Sensitive Protein 5A.The DNA of all living organisms is constantly exposed to damaging factors, and therefore a number of DNA damage repair and bypass mechanisms have evolved. DNA lesions that interfere with the replication machinery constitute a particular challenge for cells (Schröpfer et al., 2014a); if not repaired in a timely manner, such damage can result in the stalling or collapse of replication forks, which in turn can lead to cell death. Furthermore, one-sided double-strand breaks (DSBs) can occur when the replication fork encounters a single-strand break. Lesions within one DNA strand, such as alkylations or DNA intrastrand cross links, can be bypassed by postreplicative repair (PRR), a process that is best understood in yeast (Saccharomyces cerevisiae). This mechanism does not lead to repair of the lesion but prevents fatal long-lasting stalling of the replication fork. PRR can be divided into two branches: the error-prone pathway and the error-free pathway (for review, see Goodman and Woodgate, 2013; Haynes et al., 2015; Jansen et al., 2015). It is known from yeast that both branches of PRR are controlled by the Radiation sensitivity protein6 (Rad6) and Mms-Ubc13 E3 ubiquitin-conjugating enzyme complexes, which ubiquitinate the replicative processivity factor Proliferating Cellular Nuclear Antigen1. The monoubiquitination of PCNA at Lys-164 by Rad6-Rad18 initiates the error-prone pathway, whereas polyubiquitination additionally requires Mms2, Ubc13, and Rad5, and triggers the error-free PRR branch (Hoege et al., 2002; Moldovan et al., 2007; Lee and Myung, 2008). There are two possible competing models postulated for the error-free bypass of lesions at the replication fork, both of which depend on template-switch mechanisms; if the lesion concerns only one of the two sister strands, the undamaged strand can be used as the template for bypassing the lesion. One of the two models features the so-called overshoot synthesis, whereby the newly synthesized strand on the undamaged parental strand is elongated further than the strand blocked by the lesion. Regression of the replication fork then leads to the formation of a special type of four-way junction called a chicken-foot structure. This regression mechanism is thought to be accomplished by helicases, such as the RecQ helicase Bloom Syndrome Protein (BLM) in humans (Croteau et al., 2014). AtRECQ4A is the respective BLM homolog in Arabidopsis (Arabidopsis thaliana), and this enzyme has the ability to regress replication forks in vitro (Hartung et al., 2007, 2008; Schröpfer et al., 2014b). The second error-free subpathway entails invasion of the newly synthesized strand on the blocked sister chromatid into the complementary newly replicated strand on the other sister chromatid. Such a step forms a displacement loop-like structure in which synthesis over the damaged region can occur. In yeast, both error-free pathways are dependent on the multifunctional protein Rad5, which is known to recruit PRR factors and also exhibits helicase activity itself (Blastyák et al., 2007). We previously identified AtRAD5A as a functional Arabidopsis homolog of Rad5 (Chen et al., 2008). Interestingly, AtRAD5A is required for efficient repair by homologous recombination via the synthesis-dependent strand-annealing mechanism, a pathway that in some steps is related to the invasion model of PRR (Mannuss et al., 2010).The error-prone pathway is based on the function of translesion synthesis (TLS) polymerases, which promote replication through DNA lesions (Prakash et al., 2005). In a mechanism termed polymerase switch, the replicative polymerase is exchanged by such a TLS polymerase at a damaged site. After incorporation of a nucleotide opposite the damaged base by the TLS polymerase, a second polymerase switch exchanges the TLS polymerase for the replicative polymerase so that replication can proceed (Prakash and Prakash, 2002; Lehmann et al., 2007). TLS polymerases possess no 5′-3′-exonuclease activity, and therefore act in a potentially mutagenic manner. Nevertheless, depending on the damage incurred and the TLS polymerase used, damage bypass can be error free (Haracska et al., 2000; McCulloch et al., 2004).Polymerases can be divided into at least six families based on their amino acid sequences and crystal structures: A, B, C, D, X, and Y. All of them share the common structure analogous to a right hand grasping DNA with palm, finger, and thumb domains (Steitz, 1999). The amino acid sequences of the finger and thumb domains of different polymerase families are highly variable, whereas the palm domains share high similarity. The palm domain forms the largest part of the polymerase active site and contains highly conserved Asp residues that have been postulated to be involved in the catalytic activity of the enzyme (Joyce and Steitz, 1995; Steitz, 1999).Most TLS polymerases belong to the Y family of polymerases, a class of specially structured enzymes that catalyze replication over damaged templates (Ohmori et al., 2001; Sale et al., 2012). Although some polymerases of the A, B, or X family can also exhibit TLS activity, this is often not their primary function (Prakash et al., 2005). DNA Polymerase Zeta (POLζ) is a B family polymerase and consists of a DNA Polymerase Zeta subunit REV3-REV7 heterodimer, in which REV3 is the catalytic subunit with its accessory subunit and processivity factor REV7 (Nelson et al., 1996). Recent studies in yeast and human cells have shown that POLζ contains two additional subunits, Pol31 and Pol32 in yeast, orthologs to human POLD2 and POLD3, which are known to be accessory subunits of the replicative polymerase POLδ (Johnson et al., 2012; Lee et al., 2014). REV3 contains three regions that are highly conserved between organisms: an N-terminal region, a REV7 binding domain, and a B family-type polymerase domain. The polymerase domain carries the six common conserved regions, I to VI (IV-II-VI-III-I-V), of which I is the most and VI the least conserved region. The A (II), B (III), and C (I) motifs, located within regions I, II, and III (Wong et al., 1988), form the active site of the enzyme, and each harbors an essential Asp residue that coordinates two catalytic metal ions. Deficiency of REV3 in mice is embryo lethal (Bemark et al., 2000; Esposito et al., 2000), and vertebrate cells depleted in REV3 show hypersensitivity to various DNA-damaging agents, including UV and ionizing irradiation, cisplatin, MMS, and mitomycin C (MMC; Sonoda et al., 2003; Sharma and Canman, 2012). In Arabidopsis, rev3 mutants exhibit no obvious phenotype under standard growth conditions, but are hypersensitive to UV-B and gamma irradiation, MMC, MMS, and cisplatin (Sakamoto et al., 2003).Our previous work demonstrated the existence of several different pathways in Arabidopsis involved in repairing the DNA damage induced by cross-linking and methylating agents. These independent pathways are defined by the ATPase RAD5A, the helicase RECQ4A, and MMS and UV-sensitive protein81 (MUS81; Mannuss et al., 2010). The structure-specific endonuclease MUS81 together with its noncatalytic subunit (Mms4 in yeast, Eme1 in Schizosaccharomyces pombe, and MMS4 or Crossover Junction Endonuclease (EME1) in humans and plants) functions in the rescue of stalled replication forks. The enzyme is able to cleave the stalled fork at the lesion site, which leads to a one-sided DSB that is repaired by homologous recombination (HR) to restore the stalled fork (Hanada et al., 2006). We previously showed that mus81 transfer DNA (T-DNA) insertion lines in Arabidopsis are highly sensitive to treatment with MMS, cisplatin, hydroxyurea, ionizing irradiation, and MMC. We also found that AtMUS81 can form a complex with its heterologous binding partners AtEME1A or AtEME1B that is able to process intricate DNA structures, such as nicked Holliday junctions, which might also form at stalled replication forks (Hartung et al., 2006; Geuting et al., 2009).In the current study, we address whether fully functional polymerase activity is required for the repair of DNA damage induced by alkylating and cross-link-inducing agents. Moreover, we sought to clarify whether REV3 cooperates with other key factors identified in Arabidopsis in the repair of these different types of damage. Indeed, it has already been shown that AtREV3 and AtRAD5A do not cooperate in the repair of such DNA damage, confirming independent pathways of error-prone and error-free PRR in plants (Wang et al., 2011). However, as plants, animals, and yeast differ in their DNA cross-link repair machinery (e.g. Mannuss et al., 2010; Knoll et al., 2012; Dangel et al., 2014; Herrmann et al., 2015), it is of particular importance to define the role of REV3 in relation to MUS81 and RECQ4A.