scholarly journals Identification of PCNA interacting protein motifs in human DNA polymerase delta

2020 ◽  
Author(s):  
Prashant Khandagale ◽  
Shweta Thakur ◽  
Narottam Acharya
Keyword(s):  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Dna Polymerase Delta ◽  
Interacting Protein ◽  
Protein Motifs ◽  
Human Dna ◽  
Biochemical Studies ◽  
Processivity Factor ◽  
Proliferating Cell

AbstractDNA polymerase delta (Polδ) is a highly processive essential replicative DNA polymerase. In humans, Polδ holoenzyme consists of p125, p50, p68, and p12 subunits and recently, we have shown that p12 exists as a dimer. Extensive biochemical studies suggest that all the subunits of Polδ interact with the processivity factor proliferating cell nuclear antigen (PCNA) to carry out a pivotal role in genomic DNA replication. While PCNA interaction protein (PIP) motifs in p68, p50 and p12 have been mapped, the PIP in p125, the catalytic subunit of the holoenzyme, remains elusive. Therefore, in this study by using multiple approaches we have conclusively mapped a non-canonical PIP box from residues 999VGGLLAFA1008 in p125, which binds to inter domain-connecting loop of PCNA with high affinity. Collectively, including previous studies, we conclude that similar to S. cerevisiae Polδ, each of the human Polδ subunits possess motif to interact with PCNA and significantly contribute towards the processive nature of this replicative DNA polymerase.

scholarly journals Identification of PCNA-interacting protein motifs in human DNA polymerase δ

2020 ◽  
Vol 40 (4) ◽  
Author(s):  
Prashant Khandagale ◽  
Shweta Thakur ◽  
Narottam Acharya
Keyword(s):  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Protein Motif ◽  
Interacting Protein ◽  
Protein Motifs ◽  
Dna Polymerase Δ ◽  
Human Dna ◽  
Processivity Factor ◽  
Proliferating Cell

Abstract DNA polymerase δ (Polδ) is a highly processive essential replicative DNA polymerase. In humans, the Polδ holoenzyme consists of p125, p50, p68 and p12 subunits and recently, we showed that the p12 subunit exists as a dimer. Extensive biochemical studies suggest that all the subunits of Polδ interact with the processivity factor proliferating cell nuclear antigen (PCNA) to carry out a pivotal role in genomic DNA replication. While PCNA-interacting protein motif (PIP) motifs in p68, p50 and p12 have been mapped, same in p125, the catalytic subunit of the holoenzyme, remains elusive. Therefore, in the present study by using multiple approaches we have conclusively mapped a non-canonical PIP motif from residues 999VGGLLAFA1008 in p125, which binds to the inter-domain-connecting loop (IDCL) of PCNA with high affinity. Collectively, including previous studies, we conclude that similar to Saccharomyces cerevisiae Polδ, each of the human Polδ subunits possesses motif to interact with PCNA and significantly contributes toward the processive nature of this replicative DNA polymerase.

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.

Proliferating cell nuclear antigen-dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair

1994 ◽  
Vol 14 (9) ◽  
pp. 6187-6197
Author(s):  
Y Matsumoto ◽  
K Kim ◽  
D F Bogenhagen
Keyword(s):  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Dna Polymerase Delta ◽  
Polymerase Beta ◽  
Ap Sites ◽  
Base Excision ◽  
Proliferating Cell ◽  
Dependent Pathway ◽  
Ap Site

DNA damage frequently leads to the production of apurinic/apyrimidinic (AP) sites, which are presumed to be repaired through the base excision pathway. For detailed analyses of this repair mechanism, a synthetic analog of an AP site, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), has been employed in a model system. Tetrahydrofuran residues are efficiently repaired in a Xenopus laevis oocyte extract in which most repair events involve ATP-dependent incorporation of no more than four nucleotides (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989; Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 11:4441-4447, 1991). Using a series of column chromatography procedures to fractionate X. laevis ovarian extracts, we developed a reconstituted system of tetrahydrofuran repair with five fractions, three of which were purified to near homogeneity: proliferating cell nuclear antigen (PCNA), AP endonuclease, and DNA polymerase delta. This PCNA-dependent system repaired natural AP sites as well as tetrahydrofuran residues. DNA polymerase beta was able to replace DNA polymerase delta only for repair of natural AP sites in a reaction that did not require PCNA. DNA polymerase alpha did not support repair of either type of AP site. This result indicates that AP sites can be repaired by two distinct pathways, the PCNA-dependent pathway and the DNA polymerase beta-dependent pathway.

scholarly journals Proliferating cell nuclear antigen-dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair.

1994 ◽  
Vol 14 (9) ◽  
pp. 6187-6197 ◽  
Author(s):  
Y Matsumoto ◽  
K Kim ◽  
D F Bogenhagen
Keyword(s):  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Dna Polymerase Delta ◽  
Polymerase Beta ◽  
Ap Sites ◽  
Base Excision ◽  
Proliferating Cell ◽  
Dependent Pathway ◽  
Ap Site

DNA damage frequently leads to the production of apurinic/apyrimidinic (AP) sites, which are presumed to be repaired through the base excision pathway. For detailed analyses of this repair mechanism, a synthetic analog of an AP site, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), has been employed in a model system. Tetrahydrofuran residues are efficiently repaired in a Xenopus laevis oocyte extract in which most repair events involve ATP-dependent incorporation of no more than four nucleotides (Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 9:3750-3757, 1989; Y. Matsumoto and D. F. Bogenhagen, Mol. Cell. Biol. 11:4441-4447, 1991). Using a series of column chromatography procedures to fractionate X. laevis ovarian extracts, we developed a reconstituted system of tetrahydrofuran repair with five fractions, three of which were purified to near homogeneity: proliferating cell nuclear antigen (PCNA), AP endonuclease, and DNA polymerase delta. This PCNA-dependent system repaired natural AP sites as well as tetrahydrofuran residues. DNA polymerase beta was able to replace DNA polymerase delta only for repair of natural AP sites in a reaction that did not require PCNA. DNA polymerase alpha did not support repair of either type of AP site. This result indicates that AP sites can be repaired by two distinct pathways, the PCNA-dependent pathway and the DNA polymerase beta-dependent pathway.

scholarly journals Proliferating Cell Nuclear Antigen-dependent Coordination of the Biological Functions of Human DNA Polymerase ι

2004 ◽  
Vol 279 (46) ◽  
pp. 48360-48368 ◽  
Author(s):  
Antonio E. Vidal ◽  
Patricia Kannouche ◽  
Vladimir N. Podust ◽  
Wei Yang ◽  
Alan R. Lehmann ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Biological Functions ◽  
Human Dna ◽  
Proliferating Cell ◽  
Dna Polymerase Ι
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