scholarly journals Isolation and Characterization of New Proliferating Cell Nuclear Antigen (POL30) Mutator Mutants That Are Defective in DNA Mismatch Repair

2002 ◽  
Vol 22 (19) ◽  
pp. 6669-6680 ◽  
Author(s):  
Patrick J. Lau ◽  
Hernan Flores-Rozas ◽  
Richard D. Kolodner
Keyword(s):  
Amino Acid ◽  
Mismatch Repair ◽  
Proliferating Cell Nuclear Antigen ◽  
Dna Mismatch Repair ◽  
Nuclear Antigen ◽  
Dna Mismatch ◽  
Isolation And Characterization ◽  
Proliferating Cell

ABSTRACT A number of studies have suggested a role for proliferating cell nuclear antigen (PCNA) in DNA mismatch repair (MMR). However, the majority of mutations in the POL30 gene encoding PCNA that cause MMR defects also cause replication and other repair defects that contribute to the increased mutation rate caused by these mutations. Here, 20 new pol30 mutants were identified and screened for MMR and other defects, resulting in the identification of two mutations, pol30-201 and pol30-204, that appear to cause MMR defects but little if any other defects. The pol30-204 mutation altered an amino acid (C81R) in the monomer-monomer interface region and resulted in a partial general MMR defect and a defect in MSH2-MSH6 binding in vitro. The pol30-201 mutation altered an amino acid (C22Y) located on the surface of the PCNA trimer that slides over the DNA but did not cause a defect in MSH2-MSH6 binding in vitro. The pol30-201 mutation caused an intermediate mutator phenotype. However, the pol30-201 mutation caused almost a complete defect in the repair of AC and GT mispairs and only a small defect in the repair of a “+T” insertion, an effect similar to that caused by an msh6Δ mutation, indicating that pol30-201 primarily effects MSH6-dependent MMR. The chromosomal double mutant msh3-FF>AA msh6-FF>AA eliminating the conserved FF residues of the PCNA interacting motif of these proteins caused a small (<10%) defect in MMR but showed synergistic interactions with mutations in POL30, indicating that the FF>AA substitution may not eliminate PCNA interactions in vivo. These results indicate that the interaction between PCNA and MMR proteins is more complex than was previously appreciated.

scholarly journals Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair

2017 ◽  
Vol 114 (19) ◽  
pp. 4930-4935 ◽  
Author(s):  
Jochen Genschel ◽  
Lyudmila Y. Kadyrova ◽  
Ravi R. Iyer ◽  
Basanta K. Dahal ◽  
Farid A. Kadyrov ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mismatch Repair ◽  
Amino Acid Substitution ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Endonuclease Domain ◽  
Substitution Mutations ◽  
Proliferating Cell

Eukaryotic MutLα (mammalian MLH1–PMS2 heterodimer; MLH1–PMS1 in yeast) functions in early steps of mismatch repair as a latent endonuclease that requires a mismatch, MutSα/β, and DNA-loaded proliferating cell nuclear antigen (PCNA) for activation. We show here that human PCNA and MutLα interact specifically but weakly in solution to form a complex of approximately 1:1 stoichiometry that depends on PCNA interaction with the C-terminal endonuclease domain of the MutLα PMS2 subunit. Amino acid substitution mutations within a PMS2 C-terminal 721QRLIAP motif attenuate or abolish human MutLα interaction with PCNA, as well as PCNA-dependent activation of MutLα endonuclease, PCNA- and DNA-dependent activation of MutLα ATPase, and MutLα function in in vitro mismatch repair. Amino acid substitution mutations within the corresponding yeast PMS1 motif (723QKLIIP) reduce or abolish mismatch repair in vivo. Coupling of a weak allele within this motif (723AKLIIP) with an exo1Δ null mutation, which individually confer only weak mutator phenotypes, inactivates mismatch repair in the yeast cell.

scholarly journals Distinct Structural Alterations in Proliferating Cell Nuclear Antigen Block DNA Mismatch Repair

Biochemistry ◽  
2013 ◽  
Vol 52 (33) ◽  
pp. 5611-5619 ◽  
Author(s):  
Lynne M. Dieckman ◽  
Elizabeth M. Boehm ◽  
Manju M. Hingorani ◽  
M. Todd Washington
Keyword(s):  
Mismatch Repair ◽  
Proliferating Cell Nuclear Antigen ◽  
Dna Mismatch Repair ◽  
Nuclear Antigen ◽  
Structural Alterations ◽  
Dna Mismatch ◽  
Proliferating Cell

scholarly journals Evidence for Involvement of Yeast Proliferating Cell Nuclear Antigen in DNA Mismatch Repair

1996 ◽  
Vol 271 (45) ◽  
pp. 27987-27990 ◽  
Author(s):  
Robert E. Johnson ◽  
Gopala K. Kovvali ◽  
Sami N. Guzder ◽  
Neelam S. Amin ◽  
Connie Holm ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Proliferating Cell Nuclear Antigen ◽  
Dna Mismatch Repair ◽  
Nuclear Antigen ◽  
Dna Mismatch ◽  
Proliferating Cell

scholarly journals The MutSα-Proliferating Cell Nuclear Antigen Interaction in Human DNA Mismatch Repair

2008 ◽  
Vol 283 (19) ◽  
pp. 13310-13319 ◽  
Author(s):  
Ravi R. Iyer ◽  
Timothy J. Pohlhaus ◽  
Sihong Chen ◽  
Gregory L. Hura ◽  
Leonid Dzantiev ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Proliferating Cell Nuclear Antigen ◽  
Dna Mismatch Repair ◽  
Nuclear Antigen ◽  
Dna Mismatch ◽  
Human Dna ◽  
Antigen Interaction ◽  
Proliferating Cell

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells

1989 ◽  
Vol 9 (1) ◽  
pp. 57-66
Author(s):  
M Zuber ◽  
E M Tan ◽  
M Ryoji
Keyword(s):  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Living Cells ◽  
Nuclear Antigen ◽  
Plasmid Replication ◽  
Dna Polymerase Delta ◽  
Dna Polymerase Alpha ◽  
Proliferating Cell

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.

scholarly journals Deficiency in DNA mismatch repair of methylation damage is a major mutational process in cancer

2020 ◽  
Author(s):  
Hu Fang ◽  
Xiaoqiang Zhu ◽  
Jieun Oh ◽  
Jayne A. Barbour ◽  
Jason W. H. Wong
Keyword(s):  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
Dna Polymerases ◽  
Induced Mutations ◽  
Mutational Signatures ◽  
Dna Mismatch ◽  
Mmr Deficiency ◽  
Mutational Process

AbstractDNA mismatch repair (MMR) is essential for maintaining genome integrity with its deficiency predisposing to cancer1. MMR is well known for its role in the post-replicative repair of mismatched base pairs that escape proofreading by DNA polymerases following cell division2. Yet, cancer genome sequencing has revealed that MMR deficient cancers not only have high mutation burden but also harbour multiple mutational signatures3, suggesting that MMR has pleotropic effects on DNA repair. The mechanisms underlying these mutational signatures have remained unclear despite studies using a range of in vitro4,5 and in vivo6 models of MMR deficiency. Here, using mutation data from cancer genomes, we identify a previously unknown function of MMR, showing that the loss of non-canonical replication-independent MMR activity is a major mutational process in human cancers. MMR is comprised of the MutSα (MSH2/MSH6) and MutLα (MLH1/PMS2) complexes7. Cancers with deficiency of MutSα exhibit mutational signature contributions distinct from those deficient of MutLα. This disparity is attributed to mutations arising from the unrepaired deamination of 5-methylcytosine (5mC), i.e. methylation damage, as opposed to replicative errors by DNA polymerases induced mismatches. Repair of methylation damage is strongly associated with H3K36me3 chromatin but independent of binding of MBD4, a DNA glycosylase that recognise 5mC and can repair methylation damage. As H3K36me3 recruits MutSα, our results suggest that MutSα is the essential factor in mediating the repair of methylation damage. Cell line models of MMR deficiency display little evidence of 5mC deamination-induced mutations as their rapid rate of proliferation limits for the opportunity for methylation damage. We thus uncover a non-canonical role of MMR in the protection against methylation damage in non-dividing cells.

scholarly journals Stimulation of 3′→5′ Exonuclease and 3′-Phosphodiesterase Activities of Yeast Apn2 by Proliferating Cell Nuclear Antigen

2002 ◽  
Vol 22 (18) ◽  
pp. 6480-6486 ◽  
Author(s):  
Ildiko Unk ◽  
Lajos Haracska ◽  
Xavier V. Gomes ◽  
Peter M. J. Burgers ◽  
Louise Prakash ◽  
...  
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Gel Filtration ◽  
Nuclear Antigen ◽  
Binding Motif ◽  
Terminal Domain ◽  
Abasic Sites ◽  
Factor C ◽  
Proliferating Cell

ABSTRACT The Apn2 protein of Saccharomyces cerevisiae contains 3′→5′ exonuclease and 3′-phosphodiesterase activities, and these activities function in the repair of DNA strand breaks that have 3′-damaged termini and which are formed in DNA by the action of oxygen-free radicals. Apn2 also has an AP endonuclease activity and functions in the removal of abasic sites from DNA. Here, we provide evidence for the physical and functional interaction of Apn2 with proliferating cell nuclear antigen (PCNA). As indicated by gel filtration and two-hybrid studies, Apn2 interacts with PCNA both in vitro and in vivo and mutations in the consensus PCNA-binding motif of Apn2 abolish this interaction. Importantly, PCNA stimulates the 3′→5′ exonuclease and 3′-phosphodiesterase activities of Apn2. We have examined the involvement of the interdomain connector loop (IDCL) and of the carboxy-terminal domain of PCNA in Apn2 binding and found that Apn2 binds PCNA via distinct domains dependent upon whether the binding is in the absence or presence of DNA. In the absence of DNA, Apn2 binds PCNA through its IDCL domain, whereas in the presence of DNA, when PCNA has been loaded onto the template-primer junction by replication factor C, the C-terminal domain of PCNA mediates the binding.

scholarly journals Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

1989 ◽  
Vol 9 (1) ◽  
pp. 57-66 ◽  
Author(s):  
M Zuber ◽  
E M Tan ◽  
M Ryoji
Keyword(s):  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Living Cells ◽  
Nuclear Antigen ◽  
Plasmid Replication ◽  
Dna Polymerase Delta ◽  
Dna Polymerase Alpha ◽  
Proliferating Cell

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.

Characterization of the human proliferating cell nuclear antigen physico-chemical properties: aspects of double trimer stability

2006 ◽  
Vol 84 (5) ◽  
pp. 669-676 ◽  
Author(s):  
Stanislav N. Naryzhny ◽  
Leroi V. DeSouza ◽  
K.W. Michael Siu ◽  
Hoyun Lee
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Wide Spectrum ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Nuclear Antigen ◽  
Physico Chemical ◽  
The Stability ◽  
Proliferating Cell

Its toroidal structure allows the proliferating cell nuclear antigen (PCNA) to wrap around and move along the DNA fiber, thereby dramatically increasing the processivity of DNA polymerization. PCNA is also involved in the regulation of a wide spectrum of other biological functions, including epigenetic inheritance. We have recently reported that mammalian PCNA forms a double trimer complex, which may be critically important in coordinating DNA replication and other cellular functions. To gain a better understanding of the stability of PCNA complexes, we characterized the physico-chemical properties of the PCNA structure by in vivo and in vitro approaches. The data obtained by gel filtration and nondenaturing gel electrophoresis of native PCNA molecules confirm our previous observations, obtained using formaldehyde crosslinking, in which PCNA exists in the cell as a double trimer. We have also found that optimal pH (pH 6.5–7.5) is critical for the stability of the PCNA structure. The presence or absence of ATP, dithiothreitol, and Mg2+ does not affect the stability of the PCNA trimer or double trimer. However, 0.02% SDS can effectively inhibit PCNA double trimer, but not single trimer, formation. Interestingly, glycerol and ammonium sulfate significantly destabilize both PCNA trimer and double trimer structures.

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