A Small Molecule Inhibitor of Monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA) Inhibits Repair of Interstrand DNA Cross-link,Enhances DNA Double Strand Break,and Sensitizes Cancer Cells to Cisplatin

Small molecule inhibitors of proliferating cell nuclear antigen (PCNA)/PCNA interacting protein box (PIP-Box) interactions, including T2 amino alcohol (T2AA), inhibit translesion DNA synthesis. The crystal structure of PCNA in complex with T2AA revealed that T2AA bound to the surface adjacent to the subunit interface of the homotrimer of PCNA in addition to the PIP-box binding cavity. Because this site is close to Lys-164, which is monoubiquitinated by RAD18, we postulated that T2AA would affect monoubiquitinated PCNA interactions. Binding of monoubiquitinated PCNA and a purified pol η fragment containing the UBZ and PIP-box was inhibited by T2AA in vitro. T2AA decreased PCNA/pol η and PCNA/REV1 chromatin colocalization but did not inhibit PCNA monoubiquitination, suggesting that T2AA hinders interactions of pol η and REV1 with monoubiquitinated PCNA. Interstrand DNA cross-links (ICLs) are repaired by mechanisms using translesion DNA synthesis that is regulated by monoubiquitinated PCNA. T2AA significantly delayed reactivation of a reporter plasmid containing an ICL. Neutral comet analysis of cells receiving T2AA in addition to cisplatin revealed that T2AA significantly enhanced formation of DNA double strand breaks (DSBs) by cisplatin. T2AA promoted colocalized foci formation of phospho-ATM and 53BP1 and up-regulated phospho-BRCA1 in cisplatin-treated cells, suggesting that T2AA increases DSBs. When cells were treated by cisplatin and T2AA, their clonogenic survival was significantly less than that of those treated by cisplatin only. These findings show that the inhibitors of monoubiquitinated PCNA chemosensitize cells by inhibiting repair of ICLs and DSBs.     

Molecular and In Vivo Characterization of Cancer-Propagating Cells Derived from MYCN-Dependent Medulloblastoma

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. While the pathways that are deregulated in MB remain to be fully characterized, amplification and/or overexpression of the MYCN gene, which is has a critical role in cerebellar development as a regulator of neural progenitor cell fate, has been identified in several MB subgroups. Phenotypically, aberrant expression of MYCN is associated with the large-cell/anaplastic MB variant, which accounts for 5-15% of cases and is associated with aggressive disease and poor clinical outcome. To better understand the role of MYCN in MB in vitro and in vivo and to aid the development of MYCN-targeted therapeutics we established tumor-derived neurosphere cell lines from the GTML (Glt1-tTA/TRE-MYCN-Luc) genetically engineered mouse model. A fraction of GTML neurospheres were found to be growth factor independent, expressed CD133 (a marker of neural stem cells), failed to differentiate upon MYCN withdrawal and were highly tumorigenic when orthotopically implanted into the cerebellum. Principal component analyzes using single cell RNA assay data suggested that the clinical candidate aurora-A kinase inhibitor MLN8237 converts GTML neurospheres to resemble non-MYCN expressors. Correlating with this, MLN8237 significantly extended the survival of mice bearing GTML MB allografts. In summary, our results demonstrate that MYCN plays a critical role in expansion and survival of aggressive MB-propagating cells, and establish GTML neurospheres as an important resource for the development of novel therapeutic strategies.     

C-Terminal Flap Endonuclease (rad27) Mutations: Lethal Interactions With a DNA Ligase I Mutation (cdc9-p) and Suppression by Proliferating Cell Nuclear Antigen (POL30) in Saccharomyces cerevisiae

During lagging-strand DNA replication in eukaryotic cells primers are removed from Okazaki fragments by the flap endonuclease and DNA ligase I joins nascent fragments. Both enzymes are brought to the replication fork by the sliding clamp proliferating cell nuclear antigen (PCNA). To understand the relationship among these three components, we have carried out a synthetic lethal screen with cdc9-p, a DNA ligase mutation with two substitutions (F43A/F44A) in its PCNA interaction domain. We recovered the flap endonuclease mutation rad27-K325* with a stop codon at residue 325. We created two additional rad27 alleles, rad27-A358* with a stop codon at residue 358 and rad27-pX8 with substitutions of all eight residues of the PCNA interaction domain. rad27-pX8 is temperature lethal and rad27-A358* grows slowly in combination with cdc9-p. Tests of mutation avoidance, DNA repair, and compatibility with DNA repair mutations showed that rad27-K325* confers severe phenotypes similar to rad27Δ, rad27-A358* confers mild phenotypes, and rad27-pX8 confers phenotypes intermediate between the other two alleles. High-copy expression of POL30 (PCNA) suppresses the canavanine mutation rate of all the rad27 alleles, including rad27Δ. These studies show the importance of the C terminus of the flap endonuclease in DNA replication and repair and, by virtue of the initial screen, show that this portion of the enzyme helps coordinate the entry of DNA ligase during Okazaki fragment maturation.CELLULAR maintenance of genomic integrity is essential for the continued viability of all organisms. The fidelity of DNA replication has to be maintained and DNA insults have to be repaired to ensure that deleterious mutations are not passed on to progeny or cause cancerous growth. A number of cellular proteins have multiple roles in DNA replication, mutation avoidance, and repair. In Saccharomyces cerevisiae, the flap endonuclease, proliferating cell nuclear antigen (PCNA), and DNA ligase I encoded by RAD27, POL30, and CDC9, respectively, are all required for proper replication and also function to avoid mutation and to facilitate repair.The flap endonuclease, FEN-1 in humans, is a highly conserved structure-specific nuclease that has both endonuclease and 5′–3′ exonuclease activity. During lagging-strand replication these activities function to remove primers from Okazaki fragments, either by endonucleolytic cleavage of a flap made by strand displacement (Liu et al. 2004) or by sequential exonucleolytic removal of single nucleotides at the 5′ end of the primer (Murante et al. 1994).While deletion of RAD27 is not lethal to yeast cells, the rad27Δ mutant exhibits temperature-sensitive growth, is a mutator, and undergoes genomic instability (Johnson et al. 1995; Reagan et al. 1995; Tishkoff et al. 1997b; Chen and Kolodner 1999). In addition, its sensitivity to low doses of the methylating agent methylmethane sulfonate (MMS) implicates the participation of the enzyme in base excision repair (BER) (Reagan et al. 1995; Wu and Wang 1999). rad27Δ mutants have been reported to be either mildly sensitive to UV light or not sensitive to UV light (Reagan et al. 1995; Sommers et al. 1995). In the strain background that the mutant is mildly sensitive, its combination with rad2Δ yields a double mutant more sensitive than each single mutant, implying that the enzyme does not participate in RAD2-mediated nucleotide excision repair (NER) (Reagan et al. 1995). The flap endonuclease has also been implicated in double-strand break (DSB) repair by virtue of the incompatibility of rad27Δ with mutations of the DSB repair pathways (Tishkoff et al. 1997b; Symington 1998). In addition, either the yeast enzyme or its human ortholog has been shown to participate in reactions of homologous recombination, nonhomologous end joining, and telomere maintenance (Parenteau and Wellinger 1999, 2002; Wu et al. 1999; Wang et al. 2004; Kikuchi et al. 2005). Curiously, the rad27Δ mutant is not sensitive to gamma radiation but is sensitive to high doses of MMS that are thought to act as a radiomimetic agent (Reagan et al. 1995; Sommers et al. 1995).PCNA is the replicative clamp that acts as a scaffold to facilitate the loading of DNA replication and repair proteins, including DNA ligase I and the flap endonuclease to DNA (Warbrick 2000, 2006; Maga and Hubscher 2003). PCNA (POL30) is essential for cell viability, which is indicative of its central role in DNA metabolism. Biochemical characterization of its effect on the flap endonuclease shows that it stimulates its activity ∼50-fold, evidencing the productive nature of the interaction (Gomes and Burgers 2000; Tom et al. 2000; Frank et al. 2001; Stucki et al. 2001). The ability of DNA ligase to efficiently catalyze the formation of phosphodiester bonds in the DNA backbone may also be facilitated by its binding to PCNA. Tom et al. (2001) showed that, in vitro, PCNA enhances the ligation reaction 5-fold and that the stable association of DNA ligase with nicked duplex DNA requires PCNA.Both DNA ligase and the flap endonuclease bind to PCNA via their respective PCNA interactive peptide domains (PIP box). The PIP box is a conserved sequence motif of the amino acids QXXLXXFF. The PIP box fits into the interdomain connector loop (IDCL) of PCNA to provide a protein–protein interaction surface (Gomes and Burgers 2000; Chapados et al. 2004; Sakurai et al. 2005; Pascal et al. 2006). Mutations in the PIP box or the IDCL that impair the interaction of DNA ligase and the flap endonuclease to PCNA lead to genomic instability (Amin and Holm 1996; Eissenberg et al. 1997; Gary et al. 1999; Refsland and Livingston 2005; Subramanian et al. 2005). We have reported that the double mutants made by combinations of cdc9-p, rad27-p, and pol30-90—mutations with alterations of the PIP box or the IDCL in the respective proteins—have synergistic phenotypes with respect to MMS sensitivity and to trinucleotide repeat instability (Refsland and Livingston 2005). These results suggest that the two enzymes function in a concerted manner that is facilitated by PCNA.The precise nature of how PCNA coordinates the entry of the flap endonuclease and DNA ligase into the replication fork is not well understood. Biochemical and structural studies have begun to elucidate a possible ordering of these PCNA-mediated interactions. The possibility of such an ordering is underscored by the observation that DNA ligase adopts a toroidal conformation by completely encircling duplex DNA while interacting with PCNA (Pascal et al. 2004). Moreover, both PCNA and DNA ligase may be loaded onto the DNA in a mechanism utilizing the replication clamp loader replication factor C (RFC) (Levin et al. 2004; Vijayakumar et al. 2009), again suggesting a complete encirclement of the DNA by DNA ligase as well as by PCNA. PCNA and DNA ligase are similar in size and their interaction is likely to extend along the face of PCNA in a manner that would prevent other proteins such as the flap endonuclease from binding to the IDCL (Pascal et al. 2004, 2006). A biochemical study with purified yeast proteins showed that the two enzymes cannot bind simultaneously to PCNA (Subramanian et al. 2005). These studies suggest that a coordinated sequential interaction among PCNA, DNA ligase, and the flap endonuclease is important for replication and repair.Alternatively, both the flap endonuclease and DNA ligase may bind to the same molecule of PCNA. Since PCNA is a homotrimer, DNA ligase can potentially bind to one monomer while the flap endonuclease binds to another, using its extended C-terminal tail in a conformation allowing it to be tethered to PCNA concurrently with DNA ligase (Gomes and Burgers 2000; Sakurai et al. 2005). DNA ligase could also bind to PCNA in an extended conformation while the flap endonuclease cleaves the DNA. Sulfolobus solfataricus DNA ligase has been shown to have an open, extended conformation while binding to PCNA (Pascal et al. 2006). Presumably, once the flap endonuclease has removed the 5′ flap, DNA ligase acquires a closed, ring-shaped conformation to catalyze the joining of Okazaki fragments (Pascal et al. 2006).Exactly how the interaction of these enzymes with PCNA is coordinated in vivo, whether singly or concurrently, is not well understood. To further elucidate how the interaction of DNA ligase with PCNA is ordered, we performed a genetic screen to identify mutations that are synthetically lethal with cdc9-p (F44A/F35A), an allele of DNA ligase that has impaired binding to PCNA (Refsland and Livingston 2005; Subramanian et al. 2005). We postulated that genes recovered from this screen would function in DNA repair, replication, and recombination or would be involved in ordering the DNA ligase–PCNA interaction. From the screen we recovered a truncated allele of RAD27, rad27-K325*. This allele encodes a protein that lacks the PIP box and the entire C-terminal domain of the enzyme but retains the N terminus containing the nuclease activities. We have characterized this allele and compared it to two other rad27 alleles in which we have created different alterations of the C-terminal end of the flap endonuclease.     

Structure/Function Analysis of PARP-1 in Oxidative and Nitrosative Stress-Induced Monomeric ADPR Formation

Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2^nd messenger monomeric adenosine diphosphate-ribose (mADPR). Previous studies have delineated specific roles for several of PARP-1′s structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1′s BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2^nd messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.     

Down-Regulation of Filamin Ainteracting protein 1-like Is Associated with Promoter Methylation and an Invasive Phenotype in Breast,Colon, Lung and Pancreatic Cancers

Identifying key mediators of cancer cell invasion and metastasis is critical to the development of more effective cancer therapies. We previously identified Filamin A interacting protein 1-like (FILIP1L) as an important inhibitor of cell migration and invasion in ovarian cancer. FILIP1L expression was inversely correlated with the invasive potential of ovarian cancer cell lines and ovarian cancer specimens. We also demonstrated that DNA methylation in the FILIP1L promoter was a mechanism by which FILIP1L was down-regulated in ovarian cancer. In our present study, we tested this observation in other cancer histologies: breast, colon, lung and pancreatic cancers. Both mRNA and protein expression of FILIP1L were down-regulated in these cancer cells compared with their normal epithelial cells. As in ovarian cancer, DNA methylation is a mechanism by which FILIP1L is down-regulated in these cancer histologies. Methylation status of the FILIP1L promoter was inversely correlated with FILIP1L expression. Reduced methylation in the FILIP1L promoter following treatment with a DNA demethylating agent was associated with restoration of FILIP1L expression in these cancer cells. Further, FILIP1L expression was inversely correlated with the invasive potential of these cancer cells. Re-expression of FILIP1L in FILIP1L-low expressing, highly-invasive cancer cell lines resulted in inhibition of cell invasion. Correspondingly, knockdown of FILIP1L in FILIP1L-high expressing, low-invasive cancer cell lines resulted in increase of cell invasion. Overall, these findings suggest that down-regulation of FILIP1L associated with DNA methylation is related with the invasive phenotype in various cancers. Thus, modulation of FILIP1L expression has the potential to be a target for cancer therapy.     

HEK293T Cells Are Heterozygous for CCR5 Delta 32 Mutation

C-C chemokine receptor 5 (CCR5) is a receptor for chemokines and a co-receptor for HIV-1 entry into the target CD4+ cells. CCR5 delta 32 deletion is a loss-of-function mutation, resistant to HIV-1 infection. We tried to induce the CCR5 delta 32 mutation harnessing the genome editing technique, CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR and CRISPR associated protein 9, Cas9) in the commonly used cell line human embryonic kidney HEK 293T cells. Surprisingly, we found that HEK293T cells are heterozygous for CCR5 delta 32 mutation, in contrast to the wild type CCR5 cells, human acute T cell leukemia cell line Jurkat and human breast adenocarcinoma cell line MDA-MB-231 cells. This finding indicates that at least one human cell line is heterozygous for the CCR5 delta 32 mutation. We also found that in PCR amplification, wild type CCR5 DNA and mutant delta 32 DNA can form mismatched heteroduplex and move slowly in gel electrophoresis.     

Analysis of the Prevalence,Secretion and Function of a Cell Cycle-Inhibiting Factor in the Melioidosis Pathogen Burkholderia pseudomallei

Enteropathogenic and enterohaemorrhagic Escherichia coli express a cell cycle-inhibiting factor (Cif), that is injected into host cells via a Type III secretion system (T3SS) leading to arrest of cell division, delayed apoptosis and cytoskeletal rearrangements. A homologue of Cif has been identified in Burkholderia pseudomallei (CHBP; Cif homologue in B. pseudomallei; BPSS1385), which shares catalytic activity, but its prevalence, secretion and function are ill-defined. Among 43 available B. pseudomallei genome sequences, 33 genomes (76.7%) harbor the gene encoding CHBP. Western blot analysis using antiserum raised to a synthetic CHBP peptide detected CHBP in 46.6% (7/15) of clinical B. pseudomallei isolates from the endemic area. Secretion of CHBP into bacterial culture supernatant could not be detected under conditions where a known effector (BopE) was secreted in a manner dependent on the Bsa T3SS. In contrast, CHBP could be detected in U937 cells infected with B. pseudomallei by immunofluorescence microscopy and Western blotting in a manner dependent on bsaQ. Unlike E. coli Cif, CHBP was localized within the cytoplasm of B. pseudomallei-infected cells. A B. pseudomallei chbP insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared to the wild-type strain that could be restored by plasmid-mediated trans-complementation. However, there was no defect in actin-based motility or multinucleated giant cell formation by the chbP mutant. The data suggest that the level or timing of CHBP secretion differs from a known Bsa-secreted effector and that CHBP is required for selected virulence-associated phenotypes in vitro.     

H-NS Can Facilitate Specific DNA-binding by RNA Polymerase in AT-rich Gene Regulatory Regions

Extremely AT-rich DNA sequences present a challenging template for specific recognition by RNA polymerase. In bacteria, this is because the promoter −10 hexamer, the major DNA element recognised by RNA polymerase, is itself AT-rich. We show that Histone-like Nucleoid Structuring (H-NS) protein can facilitate correct recognition of a promoter by RNA polymerase in AT-rich gene regulatory regions. Thus, at the Escherichia coli ehxCABD operon, RNA polymerase is unable to distinguish between the promoter −10 element and similar overlapping sequences. This problem is resolved in native nucleoprotein because the overlapping sequences are masked by H-NS. Our work provides mechanistic insight into nucleoprotein structure and its effect on protein-DNA interactions in prokaryotic cells.     

Functional Characterisation of Anticancer Activity in the Aqueous Extract of Helicteres angustifolia L. Roots

Helicteres angustifolia L. is a shrub that forms a common ingredient of several cancer treatment recipes in traditional medicine system both in China and Laos. In order to investigate molecular mechanisms of its anticancer activity, we prepared aqueous extract of Helicteres angustifolia L. Roots (AQHAR) and performed several in vitro assays using human normal fibroblasts (TIG-3) and osteosarcoma (U2OS). We found that AQHAR caused growth arrest/apoptosis of U2OS cells in a dose-dependent manner. It showed no cytotoxicity to TIG-3 cells at doses up to 50 μg/ml. Biochemical, imaging and cell cycle analyses revealed that it induces ROS signaling and DNA damage response selectively in cancer cells. The latter showed upregulation of p53, p21 and downregulation of Cyclin B1 and phospho-Rb. Furthermore, AQHAR-induced apoptosis was mediated by increase in pro-apoptotic proteins including cleaved PARP, caspases and Bax. Anti-apoptotic protein Bcl-2 showed decrease in AQHAR-treated U2OS cells. In vivo xenograft tumor assays in nude mice revealed dose-dependent suppression of tumor growth and lung metastasis with no toxicity to the animals suggesting that AQHAR could be a potent and safe natural drug for cancer treatment.     

Molecular Characterization of a Novel Staphylococcus Aureus Surface Protein (SasC) Involved in Cell Aggregation and Biofilm Accumulation

Background

Staphylococci belong to the most important pathogens causing implant-associated infections. Colonization of the implanted medical devices by the formation of a three-dimensional structure made of bacteria and host material called biofilm is considered the most critical factor in these infections. To form a biofilm, bacteria first attach to the surface of the medical device, and then proliferate and accumulate into multilayered cell clusters. Biofilm accumulation may be mediated by polysaccharide and protein factors.

Methology/Principal Findings

The information on Staphylococcus aureus protein factors involved in biofilm accumulation is limited, therefore, we searched the S. aureus Col genome for LPXTG-motif containing potential surface proteins and chose the so far uncharacterized S. aureus surface protein C (SasC) for further investigation. The deduced SasC sequence consists of 2186 amino acids with a molecular mass of 238 kDa and has features typical of Gram-positive surface proteins, such as an N-terminal signal peptide, a C-terminal LPXTG cell wall anchorage motif, and a repeat region consisting of 17 repeats similar to the domain of unknown function 1542 (DUF1542). We heterologously expressed sasC in Staphylococcus carnosus, which led to the formation of huge cell aggregates indicative of intercellular adhesion and biofilm accumulation. To localize the domain conferring cell aggregation, we expressed two subclones of sasC encoding either the N-terminal domain including a motif that is found in various architectures (FIVAR) or 8 of the DUF1542 repeats. SasC or its N-terminal domain, but not the DUF1542 repeat region conferred production of huge cell aggregates, higher attachment to polystyrene, and enhanced biofilm formation to S. carnosus and S. aureus. SasC does not mediate binding to fibrinogen, thrombospondin-1, von Willebrand factor, or platelets as determined by flow cytometry.

Conclusions/Significance

Thus, SasC represents a novel S. aureus protein factor involved in cell aggregation and biofilm formation, which may play an important role in colonization during infection with this important pathogen.     

β-Neurexin Is a Ligand for the Staphylococcus aureus MSCRAMM SdrC

Gram-positive bacteria contain a family of surface proteins that are covalently anchored to the cell wall of the organism. These cell-wall anchored (CWA) proteins appear to play key roles in the interactions between pathogenic organisms and the host. A subfamily of the CWA has a common structural organization with multiple domains adopting characteristic IgG-like folds. The identified microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) belong to this subfamily, as does SdrC from S. aureus. However, an interactive host ligand for the putative MSCRAMM SdrC was not previously identified. We have screened a phage display peptide library and identified a peptide sequence found in β-neurexin that binds SdrC. A synthetic peptide corresponding to the identified sequence as well as a recombinant form of the β-neurexin 1 exodomain binds SdrC with high affinity and specificity. Furthermore, expression of SdrC on bacteria greatly enhances microbial adherence to cultured mammalian cells expressing β-neurexin on their surface. Taken together, our experimental results demonstrate that β-neurexin is a ligand for SdrC. This interaction involves a specific sequence located in the N-terminal region of the mammalian protein and the N₂N₃ domain of the MSCRAMM. The fact that these two proteins interact when expressed on the appropriate cells demonstrates the functionality of the interaction. Possible implications of this interaction are discussed.     

Cell Wall-Anchored Nuclease of Streptococcus sanguinis Contributes to Escape from Neutrophil Extracellular Trap-Mediated Bacteriocidal Activity

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg²⁺ and Ca²⁺ for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression.     

Properties of a Bacillus subtilis strain lacking DNA polymerase I

We have isolated a mutant of Bacillussubtilis deficient in DNA polymerase I, denominated polA42, which shows a reduced ability to repair the damage to DNA by UV radiation, MMS and mitomycin C;the ability to perform recombination is not appreciably impaired.DEAE cellulose chromatography allows the separation of polymerases I and II from the parental strain;a simple procedure is also described which allows to separate rapidly the polymerases II and III of the mutant strain. The three separated polymerases have similar catalytic properties but they can be distinguished for their sensitivity to inhibitors: PCMB inhibits polymerases II and III but not polymerase I; HPUra inhibits only polymerase III. All three enzymes are unaffected by nalidixate. The DNA synthesis occurring in cells of the polA42 strain permeabilized with toluene is inhibited by nalidixate, whereas the synthesis occurring in polA⁺ toluenized cells is unaffected by the drug. The polA gene has been mapped by transduction and localized between the phe₁₂ and argA₃ genes.     

Clustered DNA Lesions Containing 5-Formyluracil and AP Site: Repair via the BER System

Lesions in the DNA arise under ionizing irradiation conditions or various chemical oxidants as a single damage or as part of a multiply damaged site within 1–2 helical turns (clustered lesion). Here, we explored the repair opportunity of the apurinic/apyrimidinic site (AP site) composed of the clustered lesion with 5-formyluracil (5-foU) by the base excision repair (BER) proteins. We found, that if the AP site is shifted relative to the 5-foU of the opposite strand, it could be repaired primarily via the short-patch BER pathway. In this case, the cleavage efficiency of the AP site-containing DNA strand catalyzed by human apurinic/apyrimidinic endonuclease 1 (hAPE1) decreased under AP site excursion to the 3''-side relative to the lesion in the other DNA strand. DNA synthesis catalyzed by DNA polymerase lambda was more accurate in comparison to the one catalyzed by DNA polymerase beta. If the AP site was located exactly opposite 5-foU it was expected to switch the repair to the long-patch BER pathway. In this situation, human processivity factor hPCNA stimulates the process.