The Escherichia coli dinB gene encodes DNA polymerase (pol) IV, a protein involved in increasing spontaneous mutations in vivo. The protein-coding region of DINB1, the human ortholog of DNA pol IV, was fused to glutathione S-transferase and expressed in insect cells. The purified fusion protein was shown to be a templatedirected DNA polymerase that we propose to designate pol. Human pol lacks detectable 3 3 5 proofreading exonuclease activity and is not stimulated by recombinant human proliferating cell nuclear antigen in vitro. Between pH 6.5 and 8.5, human pol possesses optimal activity at 37°C over the pH range 6.5-7.5, and is insensitive to inhibition by aphidicolin, dideoxynucleotides, or NaCl up to 50 mM. Either Mg 2؉ or Mn 2؉ can satisfy a metal cofactor requirement for pol activity, with Mg 2؉ being preferred. Human pol is unable to bypass a cisplatin adduct in the template. However, pol shows limited bypass of an 2-acetylaminofluorene lesion and can incorporate dCTP or dTTP across from this lesion, suggesting that the bypass is potentially mutagenic. These results are consistent with a model in which pol acts as a specialized DNA polymerase whose possible role is to facilitate the replication of templates containing abnormal bases, or possessing structurally aberrant replication forks that inhibit normal DNA synthesis.We previously reported the cloning and characterization of the human DINB1 and mouse Dinb1 genes, mammalian orthologs of the Escherichia coli dinB gene (1) and members of the UmuC/DinB superfamily of DNA polymerases (2). Expression of the E. coli dinB gene is tightly regulated by the SOS system (3). Following exposure of E. coli cells to DNA-damaging agents such as ultraviolet (UV) radiation, induction of dinB results in enhanced spontaneous (untargeted) mutagenesis of phage DNA introduced into the bacteria subsequent to irradiation (4). Increased spontaneous mutagenesis is also observed following overexpression of dinB in cells transfected with FЈlac plasmids, with the most prevalent mutations detected being Ϫ1 frameshifts (5). Recombinant E. coli DinB protein carrying a 6-histidine tag was purified and shown to be a DNA polymerase, designated DNA pol IV of E. coli, which is devoid of detectable exonuclease activity (6). Consistent with its apparent ability to generate frameshift mutations in vivo, DNA pol IV is able to extend a misaligned primer-template in vitro, resulting in a Ϫ1 frameshift mutation (6). More recently, DNA pol IV has been shown to be unable to efficiently bypass an abasic site, thymine dimer, or 6-4 photoproduct in vitro (7). Based on these observations, it has been suggested that DNA pol IV is a specialized enzyme whose role is to negotiate sites of stalled or arrested DNA replication caused by structurally abnormal replication forks, such as those caused by slippage at repeated sequences (2, 6, 7).Human DINB1 cDNA is predicted to encode a polypeptide with a molecular mass of 99 kDa, which shares extensive amino acid sequence homology with E. coli DNA pol IV, includin...
Human polymerase (pol), the product of the human POLK (DINB1) gene, is a member of the Y superfamily of DNA polymerases that support replicative bypass of chemically modified DNA bases (Ohmori, H., Friedberg, E. C., Fuchs, R. P., Goodman, M. F., Hanaoka, F., Hinkle, D., Kunkel, T. A., Lawrence, C. W., Livneh, Z., Nohmi, T., Prakash, L., Prakash, S., Todo, T., Walker, G. C., Wang, Z., and Woodgate, R. (2001) Mol. Cell 8, 7-8; Gerlach, V. L., Aravind, L., Gotway, G., Schultz, R. A., Koonin, E. V., and Friedberg, E. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11922-11927). Pol is shown here to bypass 5,6-dihydro-5,6-dihydroxythymine (thymine glycol) generated in two different DNA substrate preparations. Pol inserts the correct base adenine opposite thymine glycol in preference to the other three bases. Additionally, the enzyme correctly extends beyond the site of the thymine glycol lesion when presented with adenine opposite thymine glycol at the primer terminus. However, steady state kinetic analysis of nucleotides incorporated opposite thymine glycol demonstrates different misincorporation rates for guanine with each of the two DNA substrates. The two substrates differ only in the relative proportions of thymine glycol stereoisomers, suggesting that pol distinguishes among stereoisomers and exhibits reduced discrimination between purines when incorporating a base opposite a 5R thymine glycol stereoisomer. When extending beyond the site of the lesion, the misincorporation rate of pol for each of the three incorrect nucleotides (adenine, guanine, and thymine) is dramatically increased. Our findings suggest a role for pol in both nonmutagenic and mutagenic bypass of oxidative damage.Many types of base damage in DNA cause structural modifications that can result in the stalling or complete arrest of high fidelity DNA synthesis during DNA replication (3, 4). However, the potential for cell death attendant on arrested DNA replication can be mitigated by a mechanism called translesion DNA synthesis (TLS) (5-7). This process effects the replicative bypass of sites of base damage, allowing high fidelity semiconservative DNA synthesis to continue. Important new insights into the biochemical mechanism of TLS have recently been gained by the discovery of a number of new DNA polymerases, all of which share the properties of limited fidelity and processivity when copying undamaged DNA, as well as a lack of 3Ј 3 5Ј proofreading exonuclease activity (1, 5-9). Multiple DNA polymerases of this class have been shown to support TLS of one or more types of base damage in vitro. In some instances, this role is supported by genetic or other biological evidence. Hence, a general theme is beginning to emerge that the redundancy for error-prone DNA polymerases in prokaryotic and especially in eukaryotic cells reflects a requirement for the bypass of multiple types of base damage that can arrest normal DNA replication (5). Recent structural studies on a number of these polymerases suggest that translesion synthesis is effected by a less ...
REV1 is a eukaryotic member of the Y-family of DNA polymerases involved in translesion DNA synthesis and genome mutagenesis. Recently, REV1 is also found to function in homologous recombination. However, it remains unclear how REV1 is recruited to the sites where homologous recombination is processed. Here, we report that loss of mammalian REV1 results in a specific defect in replication-associated gene conversion. We found that REV1 is targeted to laser-induced DNA damage stripes in a manner dependent on its ubiquitin-binding motifs, on RAD18, and on monoubiquitinated FANCD2 (FANCD2-mUb) that associates with REV1. Expression of a FANCD2-Ub chimeric protein in RAD18-depleted cells enhances REV1 assembly at laser-damaged sites, suggesting that FANCD2-mUb functions downstream of RAD18 to recruit REV1 to DNA breaks. Consistent with this suggestion we found that REV1 and FANCD2 are epistatic with respect to sensitivity to the double-strand break-inducer camptothecin. REV1 enrichment at DNA damage stripes also partially depends on BRCA1 and BRCA2, components of the FANCD2/BRCA supercomplex. Intriguingly, analogous to FANCD2-mUb and BRCA1/BRCA2, REV1 plays an unexpected role in protecting nascent replication tracts from degradation by stabilizing RAD51 filaments. Collectively these data suggest that REV1 plays multiple roles at stalled replication forks in response to replication stress.
In the yeast Saccharomyces cerevisiae, the Rad1–Rad10 protein complex participates in nucleotide excision repair (NER) and homologous recombination (HR). During HR, the Rad1–Rad10 endonuclease cleaves 3′ branches of DNA and aberrant 3′ DNA ends that are refractory to other 3′ processing enzymes. Here we show that yeast strains expressing fluorescently labeled Rad10 protein (Rad10-YFP) form foci in response to double-strand breaks (DSBs) induced by a site-specific restriction enzyme, I-SceI or by ionizing radiation (IR). Additionally, for endonuclease-induced DSBs, Rad10-YFP localization to DSB sites depends on both RAD51 and RAD52, but not MRE11 while IR-induced breaks do not require RAD51. Finally, Rad10-YFP colocalizes with Rad51-CFP and with Rad52-CFP at DSB sites, indicating a temporal overlap of Rad52, Rad51 and Rad10 functions at DSBs. These observations are consistent with a putative role of Rad10 protein in excising overhanging DNA ends after homology searching and refine the potential role(s) of the Rad1–Rad10 complex in DSB repair in yeast.
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