scholarly journals Human DNA polymerase delta requires an iron–sulfur cluster for high-fidelity DNA synthesis

2019 ◽  
Vol 2 (4) ◽  
pp. e201900321 ◽  
Author(s):  
Stanislaw K. Jozwiakowski ◽  
Sandra Kummer ◽  
Kerstin Gari
Keyword(s):  
Dna Binding ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Binding Pocket ◽  
Nuclear Antigen ◽  
Dna Polymerase Delta ◽  
Iron Sulfur Cluster ◽  
Double Stranded Dna ◽  
Iron Sulfur ◽  
Proliferating Cell

Replication of eukaryotic genomes relies on the family B DNA polymerases Pol α, Pol δ, and Pol ε. All of these enzymes coordinate an iron–sulfur (FeS) cluster, but the function of this cofactor has remained largely unclear. Here, we show that the FeS cluster in the catalytic subunit of human Pol δ is coordinated by four invariant cysteines of the C-terminal CysB motif. FeS cluster loss causes a partial destabilisation of the four-subunit enzyme, a defect in double-stranded DNA binding, and compromised polymerase and exonuclease activities. Importantly, complex stability, DNA binding, and enzymatic activities are restored in the presence of proliferating cell nuclear antigen. We further show that also more subtle changes to the FeS cluster-binding pocket that do not abolish FeS cluster binding can have repercussions on the distant exonuclease domain and render the enzyme error prone. Our data, hence, suggest that the FeS cluster in human Pol δ is an important cofactor that despite its C-terminal location, it has an impact on both DNA polymerase and exonuclease activities and can influence the fidelity of DNA synthesis.

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.

Dual mode of interaction of DNA polymerase ϵ with proliferating cell nuclear antigen in primer binding and DNA synthesis 1 1Edited by J. Karn

1999 ◽  
Vol 285 (1) ◽  
pp. 259-267 ◽  
Author(s):  
Giovanni Maga ◽  
Zophonı́as O Jónsson ◽  
Manuel Stucki ◽  
Silvio Spadari ◽  
Ulrich Hübscher
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Dual Mode ◽  
Proliferating Cell

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 Purification and characterization of a 100 kDa DNA polymerase from cauliflower inflorescence

1998 ◽  
Vol 332 (2) ◽  
pp. 557-563 ◽  
Author(s):  
Hirokazu SETO ◽  
Masami HATANAKA ◽  
Seisuke KIMURA ◽  
Masahiko OSHIGE ◽  
Yuri TSUYA ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Purification And Characterization ◽  
Synthetic Dna ◽  
Different Types ◽  
Proliferating Cell

A DNA polymerase from cauliflower (Brassica oleracea var. botrytis) inflorescence has been purified to near homogeneity through five successive column chromatographies, and temporally designated cauliflower polymerase 1. Cauliflower polymerase 1 is a monopolypeptide with a molecular mass of 100 kDa. The enzyme efficiently uses synthetic DNA homopolymers and moderately activated DNA and a synthetic RNA homopolymer as template-primers. The enzyme is strongly sensitive to dideoxythymidine triphosphate and N-ethylmaleimide, but it is insensitive to aphidicolin. It was stimulated with 250 mM KCl. Its mode of DNA synthesis is high-processive with or without proliferating-cell nuclear antigen. A 3´ → 5´ exonuclease activity is associated with cauliflower polymerase 1. The enzyme is clearly different from cauliflower mitochondrial polymerase and does not resemble the four different types of wheat DNA polymerase, designated wheat DNA polymerases A, B, CI and CII. In the present paper the role of the enzyme in plant DNA synthesis is discussed.

scholarly journals Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites.

1987 ◽  
Vol 105 (4) ◽  
pp. 1549-1554 ◽  
Author(s):  
R Bravo ◽  
H Macdonald-Bravo
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
S Phase ◽  
Nuclear Antigen ◽  
Dna Polymerase Delta ◽  
Quiescent Cells ◽  
Replication Sites ◽  
Nuclear Structures ◽  
Proliferating Cell ◽  
Two Populations

Pulse-chase experiments have revealed that cyclin, the auxiliary protein of DNA polymerase-delta, is stable during the transition from growth to quiescence in 3T3 cells. Immunoblotting together with immunofluorescence analysis has shown that the amount of cyclin after 24 h of quiescence is 30-40% of that of growing cells and that it presents a nucleoplasmic staining. Immunofluorescence studies show the existence of two populations of cyclin during the S phase, one that is nucleoplasmic as in quiescent cells and is easily extracted by detergent, and another that is associated to specific nuclear structures. By using antibromodeoxyuridine immunofluorescence to detect the sites of DNA synthesis, it was shown that the staining patterns of the replicon clusters and their order of appearance throughout the S phase are identical to those observed for cyclin. Two-dimensional gel analysis of Triton-extracted cells show that 20-30% of cyclin remains associated with the replicon clusters. This population of cyclin could not be released from the nucleus using high-salt extractions. This demonstrates that cyclin is tightly associated to the sites of DNA replication and that it must have a fundamental role in DNA synthesis in eukaryotic cells.

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