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Dynamics of human replication factors in the elongation phase of DNA replication.

Masuda Y, Suzuki M, Piao J, Gu Y, Tsurimoto T, Kamiya K - Nucleic Acids Res. (2007)

Bottom Line: Some PCNA could remain at the primer terminus during this cycle, while the remainder slides out of the primer terminus or is unloaded once pol delta has dissociated.Furthermore, we suggest that a subunit of pol delta, POLD3, plays a crucial role in the efficient recycling of PCNA during dissociation-association cycles of pol delta.Based on these observations, we propose a model for dynamic processes in elongation complexes.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan. masudayu@hiroshima-u.ac.jp

ABSTRACT
In eukaryotic cells, DNA replication is carried out by coordinated actions of many proteins, including DNA polymerase delta (pol delta), replication factor C (RFC), proliferating cell nuclear antigen (PCNA) and replication protein A. Here we describe dynamic properties of these proteins in the elongation step on a single-stranded M13 template, providing evidence that pol delta has a distributive nature over the 7 kb of the M13 template, repeating a frequent dissociation-association cycle at growing 3'-hydroxyl ends. Some PCNA could remain at the primer terminus during this cycle, while the remainder slides out of the primer terminus or is unloaded once pol delta has dissociated. RFC remains around the primer terminus through the elongation phase, and could probably hold PCNA from which pol delta has detached, or reload PCNA from solution to restart DNA synthesis. Furthermore, we suggest that a subunit of pol delta, POLD3, plays a crucial role in the efficient recycling of PCNA during dissociation-association cycles of pol delta. Based on these observations, we propose a model for dynamic processes in elongation complexes.

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Reconstitution of DNA replication with recombinant replication factors on singly primed ss mp18 DNA. (A) SDS–PAGE analysis of purified recombinant proteins. Pol δ (2.4 μg), RFC (1.5 μg), RPA (1.2 μg) and PCNA (0.8 μg) were loaded on a SDS 4–20% gradient polyacrylamide gel and stained with CBB. (B) Requirement of replication factors for synthesis of singly primed ss mp18 DNA. Reactions were carried out for 10 min under the conditions described in the Materials and Methods section or omitting one replication factor. Products were analyzed by 0.7% alkaline-agarose gel electrophoresis as described in the Materials and Methods section. Incorporation of dNMP was measured as described in the Materials and Methods section. (C) Time course of the reaction of DNA synthesis. The reaction products were analyzed by the same procedures as for (B).
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Figure 1: Reconstitution of DNA replication with recombinant replication factors on singly primed ss mp18 DNA. (A) SDS–PAGE analysis of purified recombinant proteins. Pol δ (2.4 μg), RFC (1.5 μg), RPA (1.2 μg) and PCNA (0.8 μg) were loaded on a SDS 4–20% gradient polyacrylamide gel and stained with CBB. (B) Requirement of replication factors for synthesis of singly primed ss mp18 DNA. Reactions were carried out for 10 min under the conditions described in the Materials and Methods section or omitting one replication factor. Products were analyzed by 0.7% alkaline-agarose gel electrophoresis as described in the Materials and Methods section. Incorporation of dNMP was measured as described in the Materials and Methods section. (C) Time course of the reaction of DNA synthesis. The reaction products were analyzed by the same procedures as for (B).

Mentions: We initially tried to establish procedures to purify the replication proteins (pol δ, RFC, PCNA and RPA), at quantities sufficient for detailed biochemical studies. Because it has been shown that bacterial systems are very powerful for large-scale production of PCNA and RPA as complexes (24,28,29), we developed expression systems for heterotetrameric pol δ and heteropentameric RFC in E. coli and the complexes were then purified by conventional column chromatography (Figure 1A).Figure 1.


Dynamics of human replication factors in the elongation phase of DNA replication.

Masuda Y, Suzuki M, Piao J, Gu Y, Tsurimoto T, Kamiya K - Nucleic Acids Res. (2007)

Reconstitution of DNA replication with recombinant replication factors on singly primed ss mp18 DNA. (A) SDS–PAGE analysis of purified recombinant proteins. Pol δ (2.4 μg), RFC (1.5 μg), RPA (1.2 μg) and PCNA (0.8 μg) were loaded on a SDS 4–20% gradient polyacrylamide gel and stained with CBB. (B) Requirement of replication factors for synthesis of singly primed ss mp18 DNA. Reactions were carried out for 10 min under the conditions described in the Materials and Methods section or omitting one replication factor. Products were analyzed by 0.7% alkaline-agarose gel electrophoresis as described in the Materials and Methods section. Incorporation of dNMP was measured as described in the Materials and Methods section. (C) Time course of the reaction of DNA synthesis. The reaction products were analyzed by the same procedures as for (B).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175312&req=5

Figure 1: Reconstitution of DNA replication with recombinant replication factors on singly primed ss mp18 DNA. (A) SDS–PAGE analysis of purified recombinant proteins. Pol δ (2.4 μg), RFC (1.5 μg), RPA (1.2 μg) and PCNA (0.8 μg) were loaded on a SDS 4–20% gradient polyacrylamide gel and stained with CBB. (B) Requirement of replication factors for synthesis of singly primed ss mp18 DNA. Reactions were carried out for 10 min under the conditions described in the Materials and Methods section or omitting one replication factor. Products were analyzed by 0.7% alkaline-agarose gel electrophoresis as described in the Materials and Methods section. Incorporation of dNMP was measured as described in the Materials and Methods section. (C) Time course of the reaction of DNA synthesis. The reaction products were analyzed by the same procedures as for (B).
Mentions: We initially tried to establish procedures to purify the replication proteins (pol δ, RFC, PCNA and RPA), at quantities sufficient for detailed biochemical studies. Because it has been shown that bacterial systems are very powerful for large-scale production of PCNA and RPA as complexes (24,28,29), we developed expression systems for heterotetrameric pol δ and heteropentameric RFC in E. coli and the complexes were then purified by conventional column chromatography (Figure 1A).Figure 1.

Bottom Line: Some PCNA could remain at the primer terminus during this cycle, while the remainder slides out of the primer terminus or is unloaded once pol delta has dissociated.Furthermore, we suggest that a subunit of pol delta, POLD3, plays a crucial role in the efficient recycling of PCNA during dissociation-association cycles of pol delta.Based on these observations, we propose a model for dynamic processes in elongation complexes.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan. masudayu@hiroshima-u.ac.jp

ABSTRACT
In eukaryotic cells, DNA replication is carried out by coordinated actions of many proteins, including DNA polymerase delta (pol delta), replication factor C (RFC), proliferating cell nuclear antigen (PCNA) and replication protein A. Here we describe dynamic properties of these proteins in the elongation step on a single-stranded M13 template, providing evidence that pol delta has a distributive nature over the 7 kb of the M13 template, repeating a frequent dissociation-association cycle at growing 3'-hydroxyl ends. Some PCNA could remain at the primer terminus during this cycle, while the remainder slides out of the primer terminus or is unloaded once pol delta has dissociated. RFC remains around the primer terminus through the elongation phase, and could probably hold PCNA from which pol delta has detached, or reload PCNA from solution to restart DNA synthesis. Furthermore, we suggest that a subunit of pol delta, POLD3, plays a crucial role in the efficient recycling of PCNA during dissociation-association cycles of pol delta. Based on these observations, we propose a model for dynamic processes in elongation complexes.

Show MeSH