Specific interactions of three proliferating cell nuclear antigens with replication-related proteins in Aeropyrum pernix

Proliferating cell nuclear antigen (PCNA) is a well-known multifunctional protein involved in eukaryotic and archaeal DNA transactions. The homotrimeric PCNA ring encircles double-stranded DNA within its central hole and tethers many proteins on DNA. Plural genes encoding PCNA-like proteins have been found in the genome sequence of crenarchaeal organisms . We describe here the biochemical properties of the three PCNAs, PCNA1, PCNA2 and PCNA3, from the hyperthermophilic archaeon, Aeropyrum pernix . PCNA2 can form a trimeric structure by itself, and it also forms heterotrimeric structures with PCNA1 and PCNA3. However, neither PCNA1 nor PCNA3 can form homotrimers. The DNA synthesis activity of DNA polymerase I and II, the endonuclease activity of FEN1, and the nick-sealing activity of DNA ligase were stimulated by the complex of PCNA2 and 3 or PCNA1, 2 and 3. These results suggest that the heterotrimeric PCNA at least including PCNA2 and 3 function as the clamp in the replisome. However, PCNA2 is the most abundant in the cells throughout the growth stages among the three PCNAs, and therefore, PCNA2 may perform multitasks by changing complex composition.     

Two Family B DNA Polymerases from Aeropyrum pernix, an Aerobic Hyperthermophilic Crenarchaeote

DNA polymerase activities in fractionated cell extract of Aeropyrum pernix, a hyperthermophilic crenarchaeote, were investigated. Aphidicolin-sensitive (fraction I) and aphidicolin-resistant (fraction II) activities were detected. The activity in fraction I was more heat stable than that in fraction II. Two different genes (polA and polB) encoding family B DNA polymerases were cloned from the organism by PCR using degenerated primers based on the two conserved motifs (motif A and B). The deduced amino acid sequences from their entire coding regions contained all of the motifs identified in family B DNA polymerases for 3'-->5' exonuclease and polymerase activities. The product of polA gene (Pol I) was aphidicolin resistant and heat stable up to 80 degrees C. In contrast, the product of polB gene (Pol II) was aphidicolin sensitive and stable at 95 degrees C. These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II of Pyrodictium occultum. The deduced amino acid sequence of A. pernix Pol I exhibited the highest identities to archaeal family B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II). These results provide further evidence that the subdomain Crenarchaeota has two family B DNA polymerases. Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in euryarchaeotes, which contain one family B DNA polymerase and one heterodimeric DNA polymerase of a novel family.     

DNA repair by polymerase delta in Saccharomyces cerevisiae is not controlled by the proliferating cell nuclear antigen-like Rad17/Mec3/Ddc1 complex

DNA damage activates several mechanisms such as DNA repair and cell cycle checkpoints. The Saccharomyces cerevisiae heterotrimeric checkpoint clamp consisting of the Rad17, Mec3 and Ddc1 subunits is an early response factor to DNA damage and activates checkpoints. This complex is structurally similar to the proliferating cell nuclear antigen (PCNA), which serves as a sliding clamp platform for DNA replication. Growing evidence suggests that PCNA-like complexes play a major role in DNA repair as they have been shown to interact with and stimulate several proteins, including specialized DNA polymerases. With the aim of extending our knowledge concerning the link between checkpoint activation and DNA repair, we tested the possibility of a functional interaction between the Rad17/Mec3/Ddc1 complex and the replicative DNA polymerases alpha, delta and epsilon. The analysis of sensitivity response of single and double mutants to UVC and 8-MOP + UVA-induced DNA damage suggests that the PCNA-like component Mec3p of S. cerevisiae neither relies on nor competes with the third subunit of DNA polymerase delta, Pol32p, for lesion removal. No enhanced sensitivity was observed when inactivating components of DNA polymerases alpha and epsilon in the absence of Mec3p. The hypersensitivity of pol32Delta to photoactivated 8-MOP suggests that the replicative DNA polymerase delta also participates in the repair of mono- and bi-functional DNA adducts. Repair of UVC and 8-MOP + UVA-induced DNA damage via polymerase delta thus occurs independent of the Rad17/Mec3/Ddc1 checkpoint clamp.     

Functional Interactions of a Homolog of Proliferating Cell Nuclear Antigen with DNA Polymerases in Archaea

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. We cloned the gene encoding a PCNA homolog (PfuPCNA) from an euryarchaeote, Pyrococcus furiosus, expressed it in Escherichia coli, and characterized the biochemical properties of the gene product. The protein PfuPCNA stimulated the in vitro primer extension abilities of polymerase (Pol) I and Pol II, which are the two DNA polymerases identified in this organism to date. An immunological experiment showed that PfuPCNA interacts with both Pol I and Pol II. Pol I is a single polypeptide with a sequence similar to that of family B (alpha-like) DNA polymerases, while Pol II is a heterodimer. PfuPCNA interacted with DP2, the catalytic subunit of the heterodimeric complex. These results strongly support the idea that the PCNA homolog works as a sliding clamp of DNA polymerases in P. furiosus, and the basic mechanism for the processive DNA synthesis is conserved in the domains Bacteria, Eucarya, and Archaea. The stimulatory effect of PfuPCNA on the DNA synthesis was observed by using a circular DNA template without the clamp loader (replication factor C [RFC]) in both Pol I and Pol II reactions in contrast to the case of eukaryotic organisms, which are known to require the RFC to open the ring structure of PCNA prior to loading onto a circular DNA. Because RFC homologs have been found in the archaeal genomes, they may permit more efficient stimulation of DNA synthesis by archaeal DNA polymerases in the presence of PCNA. This is the first stage in elucidating the archaeal DNA replication mechanism.     

PCNA ubiquitination-independent activation of polymerase η during somatic hypermutation and DNA damage tolerance

The generation of high affinity antibodies in B cells critically depends on translesion synthesis (TLS) polymerases that introduce mutations into immunoglobulin genes during somatic hypermutation (SHM). The majority of mutations at A/T base pairs during SHM require ubiquitination of PCNA at lysine 164 (PCNA-Ub), which activates TLS polymerases. By comparing the mutation spectra in B cells of WT, TLS polymerase η (Polη)-deficient, PCNA(K164R)-mutant, and PCNA(K164R);Polη double-mutant mice, we now find that most PCNA-Ub-independent A/T mutagenesis during SHM is mediated by Polη. In addition, upon exposure to various DNA damaging agents, PCNA(K164R) mutant cells display strongly impaired recruitment of TLS polymerases, reduced daughter strand maturation and hypersensitivity. Interestingly, compared to the single mutants, PCNA(K164R);Polη double-mutant cells are dramatically delayed in S phase progression and far more prone to cell death following UV exposure. Taken together, these data support the existence of PCNA ubiquitination-dependent and -independent activation pathways of Polη during SHM and DNA damage tolerance.     

The human checkpoint protein hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9

DNA damage activates cell cycle checkpoints that prevent progression through the cell cycle. In yeast, the DNA damage checkpoint response is regulated by a series of genes that have mammalian homologs, including rad1, rad9, hus1, and rad17. On the basis of sequence homology, yeast and human Rad1, Rad9, and Hus1 protein homologs are predicted to structurally resemble the sliding clamp PCNA. Likewise, Rad17 homologs have extensive homology with replication factor C (RFC) subunits (p36, p37, p38, p40, and p140), which form a clamp loader for PCNA. These observations predict that Rad1, Hus1, and Rad9 might interact with Rad17 as a clamp-clamp loader pair during the DNA damage response. In this report, we demonstrate that endogenous human Rad17 (hRad17) interacts with the PCNA-related checkpoint proteins hRad1, hRad9, and hHus1. Mutational analysis of hRad1 and hRad17 demonstrates that this interaction has properties similar to the interaction between RFC and PCNA, a well characterized clamp-clamp loader pair. Moreover, we show that DNA damage affects the association of hRad17 with the clamp-like checkpoint proteins. Collectively, these data provide the first experimental evidence that hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9 in manner similar to the interaction between RFC and PCNA.     

Phosphorylated Rad18 directs DNA polymerase η to sites of stalled replication

The E3 ubiquitin ligase Rad18 guides DNA Polymerase eta (Polη) to sites of replication fork stalling and mono-ubiquitinates proliferating cell nuclear antigen (PCNA) to facilitate binding of Y family trans-lesion synthesis (TLS) DNA polymerases during TLS. However, it is unclear exactly how Rad18 is regulated in response to DNA damage and how Rad18 activity is coordinated with progression through different phases of the cell cycle. Here we identify Rad18 as a novel substrate of the essential protein kinase Cdc7 (also termed Dbf4/Drf1-dependent Cdc7 kinase [DDK]). A serine cluster in the Polη-binding motif of Rad 18 is phosphorylated by DDK. Efficient association of Rad18 with Polη is dependent on DDK and is necessary for redistribution of Polη to sites of replication fork stalling. This is the first demonstration of Rad18 regulation by direct phosphorylation and provides a novel mechanism for integration of S phase progression with postreplication DNA repair to maintain genome stability.     

PCNA trimer instability inhibits translesion synthesis by DNA polymerase η and by DNA polymerase δ

Translesion synthesis (TLS), the process by which DNA polymerases replicate through DNA lesions, is the source of most DNA damage-induced mutations. Sometimes TLS is carried out by replicative polymerases that have evolved to synthesize DNA on non-damaged templates. Most of the time, however, TLS is carried out by specialized translesion polymerases that have evolved to synthesize DNA on damaged templates. TLS requires the mono-ubiquitylation of the replication accessory factor proliferating cell nuclear antigen (PCNA). PCNA and ubiquitin-modified PCNA (UbPCNA) stimulate TLS by replicative and translesion polymerases. Two mutant forms of PCNA, one with an E113G substitution and one with a G178S substitution, support normal cell growth but inhibit TLS thereby reducing mutagenesis in yeast. A re-examination of the structures of both mutant PCNA proteins revealed substantial disruptions of the subunit interface that forms the PCNA trimer. Both mutant proteins have reduced trimer stability with the G178S substitution causing a more severe defect. The mutant forms of PCNA and UbPCNA do not stimulate TLS of an abasic site by either replicative Pol δ or translesion Pol η. Normal replication by Pol η was also impacted, but normal replication by Pol δ was much less affected. These findings support a model in which reduced trimer stability causes these mutant PCNA proteins to occasionally undergo conformational changes that compromise their ability to stimulate TLS by both replicative and translesion polymerases.     

Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamp-loading complexes

The repair of damaged DNA is coupled to the completion of DNA replication by several cell cycle checkpoint proteins, including, for example, in fission yeast Rad1^Sp, Hus1^Sp, Rad9^Sp and Rad17^Sp. We have found that these four proteins are conserved with protein sequences throughout eukaryotic evolution. Using computational techniques, including fold recognition, comparative modeling and generalized sequence profiles, we have made high confidence structure predictions for the each of the Rad1, Hus1 and Rad9 protein families (Rad17^Sc, Mec3^Sc and Ddc1^Sc in budding yeast, respectively). Each of these families was found to share a common protein fold with that of PCNA, the sliding clamp protein that tethers DNA polymerase to its template. We used previously reported genetic and biochemical data for these proteins from yeast and human cells to predict a heterotrimeric PCNA-like ring structure for the functional Rad1/Rad9/Hus1 complex and to determine their exact order within it. In addition, for each individual protein family, contact regions with neighbors within the PCNA-like ring were identified. Based on a molecular model for Rad17^Sp, we concluded that members of this family, similar to the subunits of the RFC clamp-loading complex, are capable of coupling ATP binding with conformational changes required to load a sliding clamp onto DNA. This model substantiates previous findings regarding the behavior of Rad17 family proteins upon DNA damage and within the RFC complex of clamp-loading proteins.     

Proliferating cell nuclear antigen promotes translesion synthesis by DNA polymerase zeta

DNA polymerase zeta (Pol zeta), a heterodimer of Rev3 and Rev7, is essential for DNA damage provoked mutagenesis in eukaryotes. DNA polymerases that function in a processive complex with the replication clamp proliferating cell nuclear antigen (PCNA) have been shown to possess a close match to the consensus PCNA-binding motif QxxLxxFF. This consensus motif is lacking in either subunit of Pol zeta, yet its activity is stimulated by PCNA. In particular, translesion synthesis of UV damage-containing DNA is dramatically stimulated by PCNA such that translesion synthesis rates are comparable with replication rates by Pol zeta on undamaged DNA. PCNA also stimulated translesion synthesis of a model abasic site by Pol zeta. Efficient PCNA stimulation required that PCNA was prevented from sliding off the damage-containing model oligonucleotide template-primer through the use of biotin-streptavidin bumpers or other blocks. Under those experimental conditions, facile bypass of the abasic site was also detected by DNA polymerase delta or eta (Rad30). The yeast DNA damage checkpoint clamp, consisting of Rad17, Mec3, and Ddc1, and an ortholog of human 9-1-1, has been implicated in damage-induced mutagenesis. However, this checkpoint clamp did not stimulate translesion synthesis by Pol zeta or by DNA polymerase delta.     

Archaeal DNA replication: identifying the pieces to solve a puzzle.

Archaeal organisms are currently recognized as very exciting and useful experimental materials. A major challenge to molecular biologists studying the biology of Archaea is their DNA replication mechanism. Undoubtedly, a full understanding of DNA replication in Archaea requires the identification of all the proteins involved. In each of four completely sequenced genomes, only one DNA polymerase (Pol BI proposed in this review from family B enzyme) was reported. This observation suggested that either a single DNA polymerase performs the task of replicating the genome and repairing the mutations or these genomes contain other DNA polymerases that cannot be identified by amino acid sequence. Recently, a heterodimeric DNA polymerase (Pol II, or Pol D as proposed in this review) was discovered in the hyperthermophilic archaeon, Pyrococcus furiosus. The genes coding for DP1 and DP2, the subunits of this DNA polymerase, are highly conserved in the Euryarchaeota. Euryarchaeotic DP1, the small subunit of Pol II (Pol D), has sequence similarity with the small subunit of eukaryotic DNA polymerase delta. DP2 protein, the large subunit of Pol II (Pol D), seems to be a catalytic subunit. Despite possessing an excellent primer extension ability in vitro, Pol II (Pol D) may yet require accessory proteins to perform all of its functions in euryarchaeotic cells. This review summarizes our present knowledge about archaeal DNA polymerases and their relationship with those accessory proteins, which were predicted from the genome sequences.     

Somatic hypermutation of immunoglobulin genes in Rad18 knockout mice

Somatic hypermutation (SHM) of immunoglobulin (Ig) genes is triggered by the activity of activation-induced cytidine deaminase (AID). AID induces DNA lesions in variable regions of Ig genes, and error-prone DNA repair mechanisms initiated in response to these lesions introduce the mutations that characterize SHM. Error-prone DNA repair in SHM is proposed to be mediated by low-fidelity DNA polymerases such as those that mediate trans-lesion synthesis (TLS); however, the mechanism by which these enzymes are recruited to AID-induced lesions remains unclear. Proliferating cell nuclear antigen (PCNA), the sliding clamp for multiple DNA polymerases, undergoes Rad6/Rad18-dependent ubiquitination in response to DNA damage. Ubiquitinated PCNA promotes the replacement of the replicative DNA polymerase stalled at the site of a DNA lesion with a TLS polymerase. To examine the potential role of Rad18-dependent PCNA ubiquitination in SHM, we analyzed Ig gene mutations in Rad18 knockout (KO) mice immunized with T cell-dependent antigens. We found that SHM in Rad18 KO mice was similar to wild-type mice, suggesting that Rad18 is dispensable for SHM. However, residual levels of ubiquitinated PCNA were observed in Rad18 KO cells, indicating that Rad18-independent PCNA ubiquitination might play a role in SHM.     

Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi.

The complete genome sequence of the hyperthermophilic archaeon Pyrococcus abyssi revealed the presence of a family B DNA polymerase (Pol I) and a family D DNA polymerase (Pol II). To extend our knowledge about euryarchaeal DNA polymerases, we cloned the genes encoding these two enzymes and expressed them in Escherichia coli. The DNA polymerases (Pol I and Pol II) were purified to homogeneity and characterized. Pol I had a molecular mass of approximately 90 kDa, as estimated by SDS/PAGE. The optimum pH and Mg(2+) concentration of Pol I were 8.5-9.0 and 3 mm, respectively. Pol II is composed of two subunits that are encoded by two genes arranged in tandem on the P. abyssi genome. We cloned these genes and purified the Pol II DNA polymerase from an E. coli strain coexpressing the cloned genes. The optimum pH and Mg(2+) concentration of Pol II were 6.5 and 15-20 mm, respectively. Both P. abyssi Pol I and Pol II have associated 3'-->5' exonuclease activity although the exonuclease motifs usually found in DNA polymerases are absent in the archaeal family D DNA polymerase sequences. Sequence analysis has revealed that the small subunit of family D DNA polymerase and the Mre11 nucleases belong to the calcineurin-like phosphoesterase superfamily and that residues involved in catalysis and metal coordination in the Mre11 nuclease three-dimensional structure are strictly conserved in both families. One hypothesis is that the phosphoesterase domain of the small subunit is responsible for the 3'-->5' exonuclease activity of family D DNA polymerase. These results increase our understanding of euryarchaeal DNA polymerases and are of importance to push forward the complete understanding of the DNA replication in P. abyssi.     

Lysine residue 185 of Rad1 is a topological but not a functional counterpart of lysine residue 164 of PCNA

Monoubiquitylation of the homotrimeric DNA sliding clamp PCNA at lysine residue 164 (PCNA(K164)) is a highly conserved, DNA damage-inducible process that is mediated by the E2/E3 complex Rad6/Rad18. This ubiquitylation event recruits translesion synthesis (TLS) polymerases capable of replicating across damaged DNA templates. Besides PCNA, the Rad6/Rad18 complex was recently shown in yeast to ubiquitylate also 9-1-1, a heterotrimeric DNA sliding clamp composed of Rad9, Rad1, and Hus1 in a DNA damage-inducible manner. Based on the highly similar crystal structures of PCNA and 9-1-1, K185 of Rad1 (Rad1(K185)) was identified as the only topological equivalent of PCNA(K164). To investigate a potential role of posttranslational modifications of Rad1(K185) in DNA damage management, we here generated a mouse model with a conditional deletable Rad1(K185R) allele. The Rad1(K185) residue was found to be dispensable for Chk1 activation, DNA damage survival, and class switch recombination of immunoglobulin genes as well as recruitment of TLS polymerases during somatic hypermutation of immunoglobulin genes. Our data indicate that Rad1(K185) is not a functional counterpart of PCNA(K164).     

PCNA modifications for regulation of post-replication repair pathways

Stalled DNA replication forks activate specific DNA repair mechanism called post-replication repair (PRR) pathways that simply bypass DNA damage. The bypassing of DNA damage by PRR prevents prolonged stalling of DNA replication that could result in double strand breaks (DSBs). Proliferating cell nuclear antigen (PCNA) functions to initiate and choose different bypassing pathways of PRR. In yeast, DNA replication forks stalled by DNA damage induces monoubiquitination of PCNA at K164, which is catalyzed by Rad6/Rad18 complex. PCNA monoubiquitination triggers the replacement of replicative polymerase with special translesion synthesis (TLS) polymerases that are able to replicate past DNA lesions. The PCNA interaction motif and/or the ubiquitin binding motif in most TLS polymerases seem to be important for the regulation of TLS. The TLS pathway is usually error-prone because TLS polymerases have low fidelity and no proofreading activity. PCNA can also be further polyubiquitinated by Ubc13/ Mms2/Rad5 complex, which adds an ubiquitin chain onto monoubiquitinated K164 of PCNA. PCNA polyubiquitination directs a different PRR pathway known as error-free damage avoidance, which uses the newly synthesized sister chromatid as a template to bypass DNA damage presumably through template switching mechanism. Mammalian homologues of all of the yeast PRR proteins have been identified, thus PRR is well conserved throughout evolution. Mutations of some PRR genes are associated with a higher risk for cancers in mice and human patients, strongly supporting the importance of PRR as a tumor suppressor pathway.     

DNA polymerase iota and related rad30-like enzymes

Until recently, the molecular mechanisms of translesion DNA synthesis (TLS), a process whereby a damaged base is used as a template for continued replication, was poorly understood. This area of scientific research has, however, been revolutionized by the finding that proteins long implicated in TLS are, in fact, DNA polymerases. Members of this so-called UmuC/DinB/Rev1/Rad30 superfamily of polymerases have been identified in prokaryotes, eukaryotes and archaea. Biochemical studies with the highly purified polymerases reveal that some, but not all, can traverse blocking lesions in template DNA. All of them share a common feature, however, in that they exhibit low fidelity when replicating undamaged DNA. Of particular interest to us is the Rad30 subfamily of polymerases found exclusively in eukaryotes. Humans possess two Rad30 paralogs, Rad30A and Rad30B. The RAD30A gene encodes DNA polymerase eta and defects in the protein lead to the xeroderma pigmentosum variant (XP-V) phenotype in humans. Very recently RAD30B has also been shown to encode a novel DNA polymerase, designated as Pol iota. Based upon in vitro studies, it appears that Pol iota has the lowest fidelity of any eukaryotic polymerase studied to date and we speculate as to the possible cellular functions of such a remarkably error-prone DNA polymerase.     

Evidence for a Rad18-independent frameshift mutagenesis pathway in human cell-free extracts

Bypass of replication blocks by specialized DNA polymerases is crucial for cell survival but may promote mutagenesis and genome instability. To gain insight into mutagenic sub-pathways that coexist in mammalian cells, we examined N-2-acetylaminofluorene (AAF)-induced frameshift mutagenesis by means of SV40-based shuttle vectors containing a single adduct. We found that in mammalian cells, as previously observed in E. coli, modification of the third guanine of two target sequences, 5'-GGG-3' (3G) and 5'-GGCGCC-3' (NarI site), induces -1 and -2 frameshift mutations, respectively. Using an in vitro assay for translesion synthesis, we investigated the biochemical control of these events. We showed that Pol eta, but neither Pol iota nor Pol zeta, plays a major role in the frameshift bypass of the AAF adduct located in the 3G sequence. By complementing PCNA-depleted extracts with either a wild-type or a non-ubiquitinatable form of PCNA, we found that this Pol eta-mediated pathway requires Rad18 and ubiquitination of PCNA. In contrast, when the AAF adduct is located within the NarI site, TLS is only partially dependent upon Pol eta and Rad18, unravelling the existence of alternative pathways that concurrently bypass this lesion.     

c-Jun N-terminal kinase-mediated Rad18 phosphorylation facilitates Polη recruitment to stalled replication forks

The E3 ubiquitin ligase Rad18 chaperones DNA polymerase η (Polη) to sites of UV-induced DNA damage and monoubiquitinates proliferating cell nuclear antigen (PCNA), facilitating engagement of Polη with stalled replication forks and promoting translesion synthesis (TLS). It is unclear how Rad18 activities are coordinated with other elements of the DNA damage response. We show here that Ser-409 residing in the Polη-binding motif of Rad18 is phosphorylated in a checkpoint kinase 1-dependent manner in genotoxin-treated cells. Recombinant Rad18 was phosphorylated specifically at S409 by c-Jun N-terminal kinase (JNK) in vitro. In UV-treated cells, Rad18 S409 phosphorylation was inhibited by a pharmacological JNK inhibitor. Conversely, ectopic expression of JNK and its upstream kinase mitogen-activated protein kinase kinase 4 led to DNA damage-independent Rad18 S409 phosphorylation. These results identify Rad18 as a novel JNK substrate. A Rad18 mutant harboring a Ser → Ala substitution at S409 was compromised for Polη association and did not redistribute Polη to nuclear foci or promote Polη-PCNA interaction efficiently relative to wild-type Rad18. Rad18 S409A also failed to fully complement the UV sensitivity of Rad18-depleted cells. Taken together, these results show that Rad18 phosphorylation by JNK represents a novel mechanism for promoting TLS and DNA damage tolerance.     

Integrating DNA replication with trans-lesion synthesis via Cdc7

It has long been appreciated that Cdc7 is an essential protein kinase that phosphorylates Mcm2-7 helicase subunits to promote initiation of DNA replication. In addition to its well-elucidated role in DNA replication, recent studies suggest that DDK is active in genotoxin-treated cells and may mediate aspects of the DNA damage response. However, specific role(s) of DDK and its effector targets in DNA damage signaling have not been defined. A recent study from our laboratories has identified the E3 ubiquitin ligase Rad18 as novel substrate of DDK in vitro and in human cells. Rad18 plays a central role in a post-replication DNA repair pathway termed ‘Trans-Lesion Synthesis’ (TLS) by promoting recruitment of DNA Polymerase eta (Polη) and other TLS polymerases to stalled replication forks. DDK-mediated Rad18 phosphorylation promotes Rad18-Polη complex formation and facilitates Rad18-dependent recruitment of Polη to stalled replication forks. The mechanisms that regulate Rad18-dependent TLS are incompletely understood. Our study provides the first demonstration of Rad18 regulation by direct phosphorylation and defines a novel mechanism for Rad18-dependent recruitment of TLS polymerases to stalled forks. This study also demonstrates a molecular basis for integration of TLS with S-phase progression via the essential Cdc7 kinase. These findings reveal unexpected mechanistic insights to the regulation of the TLS pathway and Polη recruitment.