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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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Effect of PCNA loaded spontaneously at ends of template DNA. (A) Schematic representation of the experimental design. A primer that covered HincII site was annealed to ss mp18 DNA. HincII (10 U) was introduced into standard reaction mixtures (25 μl) under the conditions described in the Materials and Methods section except for omitting RFC. After pre-incubation for 1 min, reactions were started by addition of pol δ. (B) Time course of DNA synthesis in the absence of RFC. (C) Titration of PCNA in the reactions without RFC. Amounts of PCNA used in the titration were the same as for Figure 2 (see legend of Figure 2). Reactions were carried out for 10 min. Autoradiograms of 0.7% alkaline-agarose gels in which the newly synthesized DNA were visualized by the incorporated [α-32P]dTMP, and incorporation of dNMP were measured as described in the Materials and Methods section.
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Figure 4: Effect of PCNA loaded spontaneously at ends of template DNA. (A) Schematic representation of the experimental design. A primer that covered HincII site was annealed to ss mp18 DNA. HincII (10 U) was introduced into standard reaction mixtures (25 μl) under the conditions described in the Materials and Methods section except for omitting RFC. After pre-incubation for 1 min, reactions were started by addition of pol δ. (B) Time course of DNA synthesis in the absence of RFC. (C) Titration of PCNA in the reactions without RFC. Amounts of PCNA used in the titration were the same as for Figure 2 (see legend of Figure 2). Reactions were carried out for 10 min. Autoradiograms of 0.7% alkaline-agarose gels in which the newly synthesized DNA were visualized by the incorporated [α-32P]dTMP, and incorporation of dNMP were measured as described in the Materials and Methods section.

Mentions: It is known that PCNA is spontaneously loaded from a double-stranded end of template DNA in an RFC-independent manner, and supports elongation with pol δ (44). Therefore, the restart reactions after dissociation of PCNA observed in Figure 3C could be due to spontaneous loading of PCNA at the ends. To determine the efficiency of RFC-independent restart, ss mp18 DNA was linearized first, then subjected to reactions in the absence of RFC. In this assay, a primer covering HincII site was annealed to ss mp18 DNA (Figure 4A). HincII was introduced in standard reaction mixtures, and then the reactions were started by addition of pol δ after pre-incubation for 1 min (Figure 4A). The time course of the reaction revealed the extension rate to be much slower (Figure 4B) than that of RFC-dependent reactions (Figure 3C) and the primer ends were hardly extended beyond the pausing site around 4 kb (Figure 4B). Next, we examined effects of varying PCNA concentrations on the size distribution of the products. The results demonstrated that the length of the products is uniformly short at a low concentration of PCNA (Figure 4C), suggesting that PCNA once assembled with pol δ is not stable during elongation. These results revealed differences between RFC-dependent and -independent reactions, indicating a requirement of RFC for efficient restart after dissociation of PCNA on linearized DNA.Figure 4.


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)

Effect of PCNA loaded spontaneously at ends of template DNA. (A) Schematic representation of the experimental design. A primer that covered HincII site was annealed to ss mp18 DNA. HincII (10 U) was introduced into standard reaction mixtures (25 μl) under the conditions described in the Materials and Methods section except for omitting RFC. After pre-incubation for 1 min, reactions were started by addition of pol δ. (B) Time course of DNA synthesis in the absence of RFC. (C) Titration of PCNA in the reactions without RFC. Amounts of PCNA used in the titration were the same as for Figure 2 (see legend of Figure 2). Reactions were carried out for 10 min. Autoradiograms of 0.7% alkaline-agarose gels in which the newly synthesized DNA were visualized by the incorporated [α-32P]dTMP, and incorporation of dNMP were measured as described in the Materials and Methods section.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Effect of PCNA loaded spontaneously at ends of template DNA. (A) Schematic representation of the experimental design. A primer that covered HincII site was annealed to ss mp18 DNA. HincII (10 U) was introduced into standard reaction mixtures (25 μl) under the conditions described in the Materials and Methods section except for omitting RFC. After pre-incubation for 1 min, reactions were started by addition of pol δ. (B) Time course of DNA synthesis in the absence of RFC. (C) Titration of PCNA in the reactions without RFC. Amounts of PCNA used in the titration were the same as for Figure 2 (see legend of Figure 2). Reactions were carried out for 10 min. Autoradiograms of 0.7% alkaline-agarose gels in which the newly synthesized DNA were visualized by the incorporated [α-32P]dTMP, and incorporation of dNMP were measured as described in the Materials and Methods section.
Mentions: It is known that PCNA is spontaneously loaded from a double-stranded end of template DNA in an RFC-independent manner, and supports elongation with pol δ (44). Therefore, the restart reactions after dissociation of PCNA observed in Figure 3C could be due to spontaneous loading of PCNA at the ends. To determine the efficiency of RFC-independent restart, ss mp18 DNA was linearized first, then subjected to reactions in the absence of RFC. In this assay, a primer covering HincII site was annealed to ss mp18 DNA (Figure 4A). HincII was introduced in standard reaction mixtures, and then the reactions were started by addition of pol δ after pre-incubation for 1 min (Figure 4A). The time course of the reaction revealed the extension rate to be much slower (Figure 4B) than that of RFC-dependent reactions (Figure 3C) and the primer ends were hardly extended beyond the pausing site around 4 kb (Figure 4B). Next, we examined effects of varying PCNA concentrations on the size distribution of the products. The results demonstrated that the length of the products is uniformly short at a low concentration of PCNA (Figure 4C), suggesting that PCNA once assembled with pol δ is not stable during elongation. These results revealed differences between RFC-dependent and -independent reactions, indicating a requirement of RFC for efficient restart after dissociation of PCNA on linearized DNA.Figure 4.

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
Related in: MedlinePlus