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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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Effects on size distribution after dilution of elongation complexes. (A) Outline of the assay. At 15 s after the reaction was started by addition of pol δ under standard reaction conditions described in the Materials and methods section, 10-fold dilution was performed with pre-warmed reaction mixtures without template but containing all the protein components or omitting one or two of them. Both reaction mixtures, before and after dilution, contained [α-32P]dTTP. After a further 10 min incubation, the reaction products were analyzed by 0.7% alkaline-agarose gel electrophoresis. (B) An autoradiogram of a 0.7% alkaline-agarose gel. The indicated proteins were omitted in the dilution mixtures. In the reaction shown in lane 9, 1 mM ATP in the dilution mixture was replaced with 2.5 mM ATPγS.
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Figure 5: Effects on size distribution after dilution of elongation complexes. (A) Outline of the assay. At 15 s after the reaction was started by addition of pol δ under standard reaction conditions described in the Materials and methods section, 10-fold dilution was performed with pre-warmed reaction mixtures without template but containing all the protein components or omitting one or two of them. Both reaction mixtures, before and after dilution, contained [α-32P]dTTP. After a further 10 min incubation, the reaction products were analyzed by 0.7% alkaline-agarose gel electrophoresis. (B) An autoradiogram of a 0.7% alkaline-agarose gel. The indicated proteins were omitted in the dilution mixtures. In the reaction shown in lane 9, 1 mM ATP in the dilution mixture was replaced with 2.5 mM ATPγS.

Mentions: As schematically shown in Figure 5A, the primer–template DNA was mixed with saturating concentrations of RPA, PCNA and RFC, and then 1 min later pol δ was added for formation of replication complexes. At 15 s after the initiation complex was presumably assembled, an aliquot of the reaction mixture was diluted 10-fold either into a pre-warmed reaction mixture containing all the auxiliary proteins and pol δ, or into a similar one omitting one or two of PCNA, RFC and pol δ (Figure 5A). Then, reactions were continued for further 10 min, and the products were analyzed by alkaline-agarose gel electrophoresis (Figure 5B). Dilution of either PCNA or pol δ resulted in a decrease in the size of the products (Figure 5B, lanes 2 and 4) as compared to the complete case (Figure 5B, lane 1). This effect was more pronounced when both PCNA and pol δ were diluted together (Figure 5B, lane 7); virtually no products were detected. On the other hand, dilution of RFC exerted no influence (compare lanes 1 and 3 in Figure 5B). Furthermore, when both PCNA and RFC were diluted together, the size distribution of products was almost identical to that with dilution of PCNA alone (compare lanes 2 and 5 in Figure 5B). These results indicated that PCNA and pol δ are supplied from solution, whereas RFC is not, during the elongation. Furthermore, we noted that when both RFC and pol δ were diluted together, the product size was decreased to a much greater extent than with dilution of pol δ alone (compare lanes 4 and 6 in Figure 5B). This suggested that when reassociation of pol δ is limited, RFC is needed from solution. The importance of this observation is discussed below.Figure 5.


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)

Effects on size distribution after dilution of elongation complexes. (A) Outline of the assay. At 15 s after the reaction was started by addition of pol δ under standard reaction conditions described in the Materials and methods section, 10-fold dilution was performed with pre-warmed reaction mixtures without template but containing all the protein components or omitting one or two of them. Both reaction mixtures, before and after dilution, contained [α-32P]dTTP. After a further 10 min incubation, the reaction products were analyzed by 0.7% alkaline-agarose gel electrophoresis. (B) An autoradiogram of a 0.7% alkaline-agarose gel. The indicated proteins were omitted in the dilution mixtures. In the reaction shown in lane 9, 1 mM ATP in the dilution mixture was replaced with 2.5 mM ATPγS.
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Figure 5: Effects on size distribution after dilution of elongation complexes. (A) Outline of the assay. At 15 s after the reaction was started by addition of pol δ under standard reaction conditions described in the Materials and methods section, 10-fold dilution was performed with pre-warmed reaction mixtures without template but containing all the protein components or omitting one or two of them. Both reaction mixtures, before and after dilution, contained [α-32P]dTTP. After a further 10 min incubation, the reaction products were analyzed by 0.7% alkaline-agarose gel electrophoresis. (B) An autoradiogram of a 0.7% alkaline-agarose gel. The indicated proteins were omitted in the dilution mixtures. In the reaction shown in lane 9, 1 mM ATP in the dilution mixture was replaced with 2.5 mM ATPγS.
Mentions: As schematically shown in Figure 5A, the primer–template DNA was mixed with saturating concentrations of RPA, PCNA and RFC, and then 1 min later pol δ was added for formation of replication complexes. At 15 s after the initiation complex was presumably assembled, an aliquot of the reaction mixture was diluted 10-fold either into a pre-warmed reaction mixture containing all the auxiliary proteins and pol δ, or into a similar one omitting one or two of PCNA, RFC and pol δ (Figure 5A). Then, reactions were continued for further 10 min, and the products were analyzed by alkaline-agarose gel electrophoresis (Figure 5B). Dilution of either PCNA or pol δ resulted in a decrease in the size of the products (Figure 5B, lanes 2 and 4) as compared to the complete case (Figure 5B, lane 1). This effect was more pronounced when both PCNA and pol δ were diluted together (Figure 5B, lane 7); virtually no products were detected. On the other hand, dilution of RFC exerted no influence (compare lanes 1 and 3 in Figure 5B). Furthermore, when both PCNA and RFC were diluted together, the size distribution of products was almost identical to that with dilution of PCNA alone (compare lanes 2 and 5 in Figure 5B). These results indicated that PCNA and pol δ are supplied from solution, whereas RFC is not, during the elongation. Furthermore, we noted that when both RFC and pol δ were diluted together, the product size was decreased to a much greater extent than with dilution of pol δ alone (compare lanes 4 and 6 in Figure 5B). This suggested that when reassociation of pol δ is limited, RFC is needed from solution. The importance of this observation is discussed below.Figure 5.

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