A Mutational Analysis of the Yeast Proliferating Cell Nuclear Antigen Indicates Distinct Roles in DNA Replication and DNA Repair

Ayyagari, Rao; Impellizzeri, K. J.; Yoder, Bonita L.; Gary, Sonja L.; Burgers, Peter M.

doi:10.1128/mcb.15.8.4420

Cited by 200 publications

(218 citation statements)

References 48 publications

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“…The latter kinases normally phosphorylate the pRB which controls cell cycle transition from the G 1 to the S-phase. Both p21 WAF1/CIP1 and gadd45 have been reported to associate with the proliferating cell nuclear antigen (PCNA) that is required for DNA polymerization and presumably plays a major role in DNA repair as well as DNA replication (Ayyagari et al, 1995). Addition of puri®ed Gadd45 protein has been reported to increase the nucleotide excision repair capacity in nuclear extracts of human cancer cells indicating a direct role in the DNA repair process (Smith et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

HPV-16 oncogenes E6 and E7 are mutagenic in normal human oral keratinocytes

Han

Baluda

et al. 1997

Oncogene

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The mutation frequency of pS189 shuttle vector plasmids is higher in human oral keratinocytes (NHOK) immortalized with cloned human papillomavirus-16 (HPV-16) genome than in primary normal NHOK (NHOK). To determine whether oncoproteins E6 and E7 of HPV-16 are responsible for the higher mutation frequency of the plasmids, we measured the mutation frequency in NHOK and in NHOK expressing the HPV-16 oncogenes (E6, E7, or E6 plus E7). We also measured the mutation frequency in NHOK expressing the E6 or E7 proteins of the non-oncogenic HPV-6b. The mutation frequency, either background or N-methyl-N'-nitro-Nnitrosoguanidine (MNNG)-induced, in NHOK expressing the HPV-16 oncoproteins (E6, E7, or E6 plus E7) was signi®cantly higher than in NHOK. The HPV-16 oncogenes did not alter the nature of the MNNG-induced mutations (G : C?A : T), but increased the frequency of deletions and insertions with or without MNNG. The background or MNNG-induced mutation frequency in NHOK expressing the HPV-6b E6 or E7 proteins was the same as in NHOK. NHOK and NHOK expressing HPV6b-E6 or E7 were able to arrest the cell cycle and enhance cellular p53, p21 WAF1/CIP1 , and Gadd45 levels when exposed to MNNG, whereas NHOK expressing the HPV-16 E6 oncogene did not demonstrate. NHOK expressing HPV-16 E7 were able to enhance cellular p53, p21 WAF1/CIP1 , and Gadd45 levels, but failed to arrest cell cycle progression when exposed to MNNG. These data indicate that HPV-16 E6 and E7 oncogenes are mutagenic in human oral keratinocytes and enhance the mutagenic e ect of MNNG. However, the E6 and E7 proteins of the`low risk' HPV-6b did not demonstrate such an ability.

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Section: Discussionmentioning

confidence: 99%

HPV-16 oncogenes E6 and E7 are mutagenic in normal human oral keratinocytes

Han

Baluda

et al. 1997

Oncogene

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“…1E, lane 4). In vivo, the pol30-52 allele shows defects in DNA replication, DNA repair, and mutagenesis (30). The damage avoidance pathway of postreplication DNA repair is activated by Lys-63-linked polyubiquitination of monoubiquitinated PCNA in a Rad5͞Mms2͞Ubc13-dependent manner (1).…”

Section: Resultsmentioning

confidence: 99%

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1

Garg

Burgers

2005

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

223

254

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In response to DNA damage, the Rad6͞Rad18 ubiquitin-conjugating complex monoubiquitinates the replication clamp proliferating cell nuclear antigen (PCNA) at Lys-164. Although ubiquitination of PCNA is recognized as an essential step in initiating postreplication repair, the mechanistic relevance of this modification has remained elusive. Here, we describe a robust in vitro system that ubiquitinates yeast PCNA specifically on Lys-164. Significantly, only those PCNA clamps that are appropriately loaded around effector DNA by its loader, replication factor C, are ubiquitinated. This observation suggests that, in vitro, only PCNA present at stalled replication forks is ubiquitinated. Ubiquitinated PCNA displays the same replicative functions as unmodified PCNA. These functions include loading onto DNA by replication factor C, as well as Okazaki fragment synthesis and maturation by the PCNA-coordinated actions of DNA polymerase ␦, the flap endonuclease FEN1, and DNA ligase I. However, whereas the activity of DNA polymerase remains unaffected by ubiquitination of PCNA, ubiquitinated PCNA specifically activates two key enzymes in translesion synthesis: DNA polymerase , the yeast Xeroderma pigmentosum ortholog, and Rev1, a deoxycytidyl transferase that functions in organizing the mutagenic DNA replication machinery. We propose that ubiquitination of PCNA increases its functionality as a sliding clamp to promote mutagenic DNA replication.DNA replication ͉ postreplication repair ͉ translesion synthesis ͉ ubiquitination ͉ yeast M odification of the replication clamp proliferating cell nuclear antigen (PCNA) has recently emerged as an important regulatory switch during DNA replication and DNA damage response. The critical site of modification on PCNA is Lys-164, and either sumoylation or ubiquitination at this residue can occur (1, 2). Although sumoylation at Lys-164 is proposed to be associated with normal replicative functions, monoubiquitination of this residue by the Rad6͞Rad18 ubiquitin E2͞E3 complex channels DNA damage into the postreplication DNA repair (PRR) pathway (1,3,4). The PRR pathway is comprised of two error-prone branches involving translesion synthesis (TLS) by error-prone DNA polymerases, and an error-free damage avoidance branch that proceeds by fork regression and template switching (5, 6). The latter branch depends on further modification of monoubiquitinated PCNA involving Rad5 and Mms2͞Ubc13 E2͞E3 catalyzed formation of polyubiquitin chains via an unusual Lys-63 ubiquitin linkage (1).Replicative DNA polymerases of the B-family possess a very restrictive active site, which promotes high-fidelity DNA replication but inhibits bypass synthesis of many DNA lesions (5,7,8). On the other hand, Y-family DNA polymerases are particularly adapted to the bypass of DNA lesions because of a more open active site. For instance, DNA polymerase (Pol ), the ortholog of the human Xeroderma pigmentosum protein, can accommodate a cis-syn pyrimidine dimer in its active site allowing facile damage bypass (9). TLS in yeast ...

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“…Yeast Polδ variants and replication protein A (RPA) were purified as described previously (30,85). Yeast proliferating cell nuclear antigen (PCNA) was purified using DEAE-Sepharose, S-Sepharose, Mono-Q, and hydroxyapatite column chromatography using the methods described by Ayyagari et al (86) and Fien and Stillman (87). Replication factor C (RFC) was kindly provided by Peter Burgers.…”

Section: Methodsmentioning

confidence: 99%

Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity

Mertz

Sharma²,

Chabes

et al. 2015

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

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Defects in DNA polymerases δ (Polδ) and e (Pole) cause hereditary colorectal cancer and have been implicated in the etiology of some sporadic colorectal and endometrial tumors. We previously reported that the yeast pol3-R696W allele mimicking a human cancer-associated variant, POLD1-R689W, causes a catastrophic increase in spontaneous mutagenesis. Here, we describe the mechanism of this extraordinary mutator effect. We found that the mutation rate increased synergistically when the R696W mutation was combined with defects in Polδ proofreading or mismatch repair, indicating that pathways correcting DNA replication errors are not compromised in pol3-R696W mutants. DNA synthesis by purified Polδ-R696W was error-prone, but not to the extent that could account for the unprecedented mutator phenotype of pol3-R696W strains. In a search for cellular factors that augment the mutagenic potential of Polδ-R696W, we discovered that pol3-R696W causes S-phase checkpoint-dependent elevation of dNTP pools. Abrogating this elevation by strategic mutations in dNTP metabolism genes eliminated the mutator effect of pol3-R696W, whereas restoration of high intracellular dNTP levels restored the mutator phenotype. Further, the use of dNTP concentrations present in pol3-R696W cells for in vitro DNA synthesis greatly decreased the fidelity of Polδ-R696W and produced a mutation spectrum strikingly similar to the spectrum observed in vivo. The results support a model in which (i) faulty synthesis by Polδ-R696W leads to a checkpoint-dependent increase in dNTP levels and (ii) this increase mediates the hypermutator effect of Polδ-R696W by facilitating the extension of mismatched primer termini it creates and by promoting further errors that continue to fuel the mutagenic pathway.DNA polymerase δ | colon cancer | dNTP pools | mutagenesis | DNA replication fidelity W hen functioning properly, DNA replication is phenomenally accurate, making ∼10 −10 mutations per base pair during each replication cycle (1). In respect to human biology, it means that, on average, less than one mutation occurs each time the human genome is replicated. This amazing exactitude is contingent upon the serial action of DNA polymerase selectivity, exonucleolytic proofreading, and DNA mismatch repair (MMR). MMR defects increase spontaneous mutagenesis in numerous model systems (2) and give rise to cancer in mice (3). In humans, inherited mutations in MMR genes predispose to colorectal cancer (CRC) in Lynch syndrome (4, 5). Additionally, MMR genes are inactivated via hypermethylation in ∼15% of sporadic CRC, endometrial cancer (EC), and gastric cancer (6).Like defects in MMR, mutations that decrease the base selectivity or proofreading activity of replicative DNA polymerases elevate spontaneous mutagenesis in eukaryotic cells (7-14) and cancer incidence in mice (15)(16)(17)(18). Germline mutations affecting the exonuclease domains of DNA polymerases δ (Polδ) and e (Pole) cause hereditary CRC (19), and somatic changes in the exonuclease domain of Pole were found in sporad...

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A Mutational Analysis of the Yeast Proliferating Cell Nuclear Antigen Indicates Distinct Roles in DNA Replication and DNA Repair

Cited by 200 publications

References 48 publications

HPV-16 oncogenes E6 and E7 are mutagenic in normal human oral keratinocytes

HPV-16 oncogenes E6 and E7 are mutagenic in normal human oral keratinocytes

Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1

Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity

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