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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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Amounts of PCNA loaded on DNA during elongation. (A) Outline of the assay. DNA was attached to magnetic beads via biotin–streptavidin linkage. The reactions were carried out for 10 min under the conditions described in the Materials and Methods section except for the amounts of pol δ (33 ng) and pol δ* (140 ng). (B) Western analysis. Chemiluminescence signals were detected with a CCD camera and quantified with reference to a standard curve for PCNA in the same blot.
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Figure 7: Amounts of PCNA loaded on DNA during elongation. (A) Outline of the assay. DNA was attached to magnetic beads via biotin–streptavidin linkage. The reactions were carried out for 10 min under the conditions described in the Materials and Methods section except for the amounts of pol δ (33 ng) and pol δ* (140 ng). (B) Western analysis. Chemiluminescence signals were detected with a CCD camera and quantified with reference to a standard curve for PCNA in the same blot.

Mentions: Since an excess PCNA was required for efficient DNA synthesis with pol δ* (Figure 6E), we considered whether PCNA might accumulate on DNA during elongation. If so, the frequency of sliding back to the primer terminus would increase and an increase in the local concentration of PCNA would help interactions with pol δ* at the primer terminus. To measure the amount of PCNA loaded on DNA directly, a primer containing an extended 5′ tail with one biotin molecule was annealed to ss mp18 DNA (Figure 7A). The primed ss mp18 DNA was then attached to magnetic beads and DNA replication reactions were carried out under standard reaction conditions (Figure 7A). The 5′ tail did not exert any influence on DNA synthesis (data not shown). In the reactions, 33 ng of pol δ and 140 ng of pol δ* were used since these amounts lead to equivalent efficiency of DNA synthesis (∼40 pmol of the incorporation of dNMP) and to the same size distribution of products (compare lane 3 of Figure 2C with lane 7 of Figure 6C). After reactions for 10 min, PCNA bound to beads was detected by western analysis (Figure 7B). In this assay, non-specific association of PCNA with beads or DNA was detected (Figure 7B, lane 2). When RFC was introduced into the reaction, an increase of binding of PCNA was observed. The increased amount of PCNA (difference between lanes 2 and 3 in Figure 7B) was 40 fmol, which was equivalent to that of primer template (33 fmol), suggesting specific loading to the primer terminus. Introduction of pol δ increased the amount of PCNA only slightly (Figure 7B, lane 4). The majority of PCNA was dissociated by introduction of HincII with a decreasing signal to background level (Figure 7B, lane 5), again indicating specific loading on the DNA. When pol δ* was used instead of pol δ, excessive accumulation of PCNA was unexpectedly not observed (Figure 7B, lane 6), suggesting that we cannot attribute the entire distributive nature of PCNA on the reaction with pol δ* to only the decreasing affinity between PCNA and pol δ*. We therefore considered the possibility that loading and unloading of PCNA is equilibrated in both pol δ and pol δ* cases, and importantly, could be accelerated in the reaction with pol δ*.Figure 7.


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)

Amounts of PCNA loaded on DNA during elongation. (A) Outline of the assay. DNA was attached to magnetic beads via biotin–streptavidin linkage. The reactions were carried out for 10 min under the conditions described in the Materials and Methods section except for the amounts of pol δ (33 ng) and pol δ* (140 ng). (B) Western analysis. Chemiluminescence signals were detected with a CCD camera and quantified with reference to a standard curve for PCNA in the same blot.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2175312&req=5

Figure 7: Amounts of PCNA loaded on DNA during elongation. (A) Outline of the assay. DNA was attached to magnetic beads via biotin–streptavidin linkage. The reactions were carried out for 10 min under the conditions described in the Materials and Methods section except for the amounts of pol δ (33 ng) and pol δ* (140 ng). (B) Western analysis. Chemiluminescence signals were detected with a CCD camera and quantified with reference to a standard curve for PCNA in the same blot.
Mentions: Since an excess PCNA was required for efficient DNA synthesis with pol δ* (Figure 6E), we considered whether PCNA might accumulate on DNA during elongation. If so, the frequency of sliding back to the primer terminus would increase and an increase in the local concentration of PCNA would help interactions with pol δ* at the primer terminus. To measure the amount of PCNA loaded on DNA directly, a primer containing an extended 5′ tail with one biotin molecule was annealed to ss mp18 DNA (Figure 7A). The primed ss mp18 DNA was then attached to magnetic beads and DNA replication reactions were carried out under standard reaction conditions (Figure 7A). The 5′ tail did not exert any influence on DNA synthesis (data not shown). In the reactions, 33 ng of pol δ and 140 ng of pol δ* were used since these amounts lead to equivalent efficiency of DNA synthesis (∼40 pmol of the incorporation of dNMP) and to the same size distribution of products (compare lane 3 of Figure 2C with lane 7 of Figure 6C). After reactions for 10 min, PCNA bound to beads was detected by western analysis (Figure 7B). In this assay, non-specific association of PCNA with beads or DNA was detected (Figure 7B, lane 2). When RFC was introduced into the reaction, an increase of binding of PCNA was observed. The increased amount of PCNA (difference between lanes 2 and 3 in Figure 7B) was 40 fmol, which was equivalent to that of primer template (33 fmol), suggesting specific loading to the primer terminus. Introduction of pol δ increased the amount of PCNA only slightly (Figure 7B, lane 4). The majority of PCNA was dissociated by introduction of HincII with a decreasing signal to background level (Figure 7B, lane 5), again indicating specific loading on the DNA. When pol δ* was used instead of pol δ, excessive accumulation of PCNA was unexpectedly not observed (Figure 7B, lane 6), suggesting that we cannot attribute the entire distributive nature of PCNA on the reaction with pol δ* to only the decreasing affinity between PCNA and pol δ*. We therefore considered the possibility that loading and unloading of PCNA is equilibrated in both pol δ and pol δ* cases, and importantly, could be accelerated in the reaction with pol δ*.Figure 7.

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