Proliferating cell nuclear antigen promotes DNA synthesis past template lesions by mammalian DNA polymerase δ

Mozzherin, Dmitry Ju.; Shibutani, Shinya; Tan, Cheng‐Keat; Downey, Kathleen M.; Fisher, Paul A.

doi:10.1073/pnas.94.12.6126

Cited by 98 publications

(100 citation statements)

References 66 publications

(35 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We therefore examined whether purified human PCNA, a sliding clamp known to stimulate the processivity of the replicative DNA polymerases pol␦ and pol⑀ (18 -20), increases the extent of DNA synthesis by GST/pol. The ability of PCNA to stimulate the activity of pol␦ on short oligonucleotide-derived primer-templates has been observed previously (21). As shown in Fig.…”

Section: Human Dinb1mentioning

confidence: 49%

Purification and Characterization of polκ, a DNA Polymerase Encoded by the Human DINB1 Gene

Gerlach¹,

Feaver²,

Fischhaber³

et al. 2001

Journal of Biological Chemistry

116

103

View full text Add to dashboard Cite

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...

show abstract

Section: Human Dinb1mentioning

confidence: 49%

Purification and Characterization of polκ, a DNA Polymerase Encoded by the Human DINB1 Gene

Gerlach¹,

Feaver²,

Fischhaber³

et al. 2001

Journal of Biological Chemistry

116

103

View full text Add to dashboard Cite

show abstract

“…In vitro bypass studies with purified mammalian DNA polymerases have shown that most polymerases can bypass abasic sites, at least under certain conditions (11,24,(50)(51)(52)(53)(54)(55). This underscores the need for in vivo TLR data to determine the physiological role of specific DNA polymerases in bypass of a particular lesion.…”

Section: Discussionmentioning

confidence: 99%

Quantitative measurement of translesion replication in human cells: Evidence for bypass of abasic sites by a replicative DNA polymerase

Avkin

Adar

Blander

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

109

104

View full text Add to dashboard Cite

Mutations in oncogenes and tumor suppressor genes are critical in the development of cancer. A major pathway for the formation of mutations is the replication of unrepaired DNA lesions. To better understand the mechanism of translesion replication (TLR) in mammals, a quantitative assay for TLR in cultured cells was developed. The assay is based on the transient transfection of cultured cells with a gapped plasmid, carrying a site-specific lesion in the gap region. Filling in of the gap by TLR is assayed in a subsequent bioassay, by the ability of the plasmid extracted from the cells, to transform an Escherichia coli indicator strain. Using this method it was found that TLR through a synthetic abasic site in the adenocarcinoma H1299, the osteogenic sarcoma Saos-2, the prostate carcinoma PC3, and the hepatoma Hep3B cell lines occurred with efficiencies of 92 ؎ 6%, 32 ؎ 2%, 72 ؎ 4%, and 26 ؎ 3%, respectively. DNA sequence analysis showed that 85% of the bypass events in H1299 cells involved insertion of dAMP opposite the synthetic abasic site. Addition of aphidicolin, an inhibitor of DNA polymerases ␣, ␦, and , caused a 4.4-fold inhibition of bypass. Analysis of two XP-V cell lines, defective in DNA polymerase , showed bypass of 89%, indicating that polymerase is not essential for bypass of abasic sites. These results suggest that in human cells bypass of abasic sites does not require the bypass-specific DNA polymerase , but it does require at least one of the replicative DNA polymerases, ␣, ␦, or . The quantitative TLR assay is expected to be useful in the molecular analysis of lesion bypass in a large variety of cultured mammalian cells.T he formation of mutations is a key event in several important biological phenomena, most notably cancer and evolution. A major mechanism for the formation of mutations is the replication of DNA lesions that have escaped DNA repair (1). For many years the molecular mechanism of this process, termed translesion replication (TLR), translesion synthesis, or lesion bypass, was not clear. Recently it was discovered that the main enzyme responsible for carrying out lesion bypass is a novel type of DNA polymerase, specialized for replicating across damaged sites in DNA. Such DNA polymerases were found in organisms ranging from Escherichia coli (DNA polymerase V; refs. 2 and 3) to humans (e.g., DNA polymerase ; refs. 4 and 5). These novel DNA polymerases comprise the new Y superfamily of DNA polymerases (6). Remarkably, humans contain four members of the Y family: DNA polymerases (4, 5), (7,8), and (9-12) and hREV1, which has dCMP transferase activity (13,14). In addition, humans are likely to have an additional polymerase involved in TLR, polymerase , product of the hREV3 and hREV7 genes (refs. 15 and 16; for review see refs. 17-23).Despite the importance of TLR mechanisms in understanding the role of mutagenesis in cancer, and despite the progress made with the discovery of Y family DNA polymerases, little is known about the mechanism of lesion bypass in mammalian cells. This is partl...

show abstract

“…PCNA is a processivity factor of DNA polymerase d (pol d), an enzyme involved in both DNA replication and NER (Kelman, 1997). Excess PCNA has been shown to promote DNA synthesis past template lesions (Mozzherin et al, 1996(Mozzherin et al, , 1997. Therefore, increased PCNA expression induced by Tax appears to suppress NER and allow replication of damaged DNA, providing a basis for the introduction of mutations into HTLV-I infected cells.…”

Section: Effect Of Tax On Cellular Dna Repairmentioning

confidence: 99%

Impact of HTLV-I Tax on cell cycle progression and the cellular DNA damage repair response

2005

View full text Add to dashboard Cite

Proliferating cell nuclear antigen promotes DNA synthesis past template lesions by mammalian DNA polymerase δ

Cited by 98 publications

References 66 publications

Purification and Characterization of polκ, a DNA Polymerase Encoded by the Human DINB1 Gene

Purification and Characterization of polκ, a DNA Polymerase Encoded by the Human DINB1 Gene

Quantitative measurement of translesion replication in human cells: Evidence for bypass of abasic sites by a replicative DNA polymerase

Impact of HTLV-I Tax on cell cycle progression and the cellular DNA damage repair response

Contact Info

Product

Resources

About