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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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Dynamics of replication factors in the elongation phase of DNA synthesis with pol δ*. (A) SDS–PAGE analysis of purified recombinant proteins. Pol δ* (1.9 μg) and POLD3 (0.5 μg) were loaded on a SDS 4–20% gradient polyacrylamide gel and stained with CBB. (B) Reconstitution of pol δ with POLD3 and pol δ*. Reactions were carried out for 10 min under the conditions described in the Materials and Methods section except for pol δ* (70 ng) or pol δ (90 ng) in the absence (−) or presence (+) of POLD3 (20 ng). (C–E) Titration of pol δ* (C), RFC (D) and PCNA (E). Amounts of pol δ* were 0 ng (lane 1), 4.3 ng (lane 2), 17 ng (lane 3), 35 ng (lane 4), 70 ng (lane 5), 100 ng (lane 6), and 140 ng (lane 7). Amounts of RFC used in the titration were 0 ng (lane 1), 2.3 ng (lane 2), 4.7 ng, (lane 3), 9.4 ng (lane 4), 19 ng (lane 5), 38 ng (lane 6) and 75 ng (lane 7). Amounts of PCNA used in titration were 0 ng (lane 1), 5.4 ng (lane 2), 11 ng (lane 3), 22 ng (lane 4), 43 ng (lane 5), 86 ng (lane 6) and 129 ng (lane 7). (F) Titration of PCNA in the presence of HincII. Amount of PCNA is same as (E). Reactions in (C) were carried out for 10 min under the conditions described in the Materials and Methods section. Reactions in (D–F) were carried out for 10 min under the conditions described in the Materials and Methods section except for the amount of pol δ* (140 ng). Products were analyzed by 0.7% alkaline-agarose gel electrophoresis and incorporation of dNMP were measured as described.
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Figure 6: Dynamics of replication factors in the elongation phase of DNA synthesis with pol δ*. (A) SDS–PAGE analysis of purified recombinant proteins. Pol δ* (1.9 μg) and POLD3 (0.5 μg) were loaded on a SDS 4–20% gradient polyacrylamide gel and stained with CBB. (B) Reconstitution of pol δ with POLD3 and pol δ*. Reactions were carried out for 10 min under the conditions described in the Materials and Methods section except for pol δ* (70 ng) or pol δ (90 ng) in the absence (−) or presence (+) of POLD3 (20 ng). (C–E) Titration of pol δ* (C), RFC (D) and PCNA (E). Amounts of pol δ* were 0 ng (lane 1), 4.3 ng (lane 2), 17 ng (lane 3), 35 ng (lane 4), 70 ng (lane 5), 100 ng (lane 6), and 140 ng (lane 7). Amounts of RFC used in the titration were 0 ng (lane 1), 2.3 ng (lane 2), 4.7 ng, (lane 3), 9.4 ng (lane 4), 19 ng (lane 5), 38 ng (lane 6) and 75 ng (lane 7). Amounts of PCNA used in titration were 0 ng (lane 1), 5.4 ng (lane 2), 11 ng (lane 3), 22 ng (lane 4), 43 ng (lane 5), 86 ng (lane 6) and 129 ng (lane 7). (F) Titration of PCNA in the presence of HincII. Amount of PCNA is same as (E). Reactions in (C) were carried out for 10 min under the conditions described in the Materials and Methods section. Reactions in (D–F) were carried out for 10 min under the conditions described in the Materials and Methods section except for the amount of pol δ* (140 ng). Products were analyzed by 0.7% alkaline-agarose gel electrophoresis and incorporation of dNMP were measured as described.

Mentions: The role of the 66 kDa subunit, POLD3, of pol δ in the dynamic processes involved in elongation, and the biochemical properties of subassembly (pol δ*) lacking the POLD3 subunit are of great interest. First, we examined the efficiency of DNA synthesis of human pol δ* using purified proteins (Figure 6A). A comparison of activities with equivalent amounts of pol δ* and pol δ demonstrated inefficiency of pol δ* under the standard reaction conditions with singly primed ss mp18 DNA (Figure 6B), decrease and heterogeneity in length of the products being observed with emphasized pausing sites (Figure 6B and C). The shorter products were shifted to longer ones at higher concentrations of pol δ* (Figure 6C), as with pol δ (Figure 2C). When the missing subunit, POLD3, was introduced into the reaction with pol δ*, the activity was restored to the level with pol δ (Figure 6B), indicating that the lower activity of pol δ* was due to the missing function of POLD3 subunit, rather than denaturation of proteins caused by incomplete assembly. The evidence presented here is consistent with reports for yeast counterparts (30,45) and human pol δ (36,46).Figure 6.


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)

Dynamics of replication factors in the elongation phase of DNA synthesis with pol δ*. (A) SDS–PAGE analysis of purified recombinant proteins. Pol δ* (1.9 μg) and POLD3 (0.5 μg) were loaded on a SDS 4–20% gradient polyacrylamide gel and stained with CBB. (B) Reconstitution of pol δ with POLD3 and pol δ*. Reactions were carried out for 10 min under the conditions described in the Materials and Methods section except for pol δ* (70 ng) or pol δ (90 ng) in the absence (−) or presence (+) of POLD3 (20 ng). (C–E) Titration of pol δ* (C), RFC (D) and PCNA (E). Amounts of pol δ* were 0 ng (lane 1), 4.3 ng (lane 2), 17 ng (lane 3), 35 ng (lane 4), 70 ng (lane 5), 100 ng (lane 6), and 140 ng (lane 7). Amounts of RFC used in the titration were 0 ng (lane 1), 2.3 ng (lane 2), 4.7 ng, (lane 3), 9.4 ng (lane 4), 19 ng (lane 5), 38 ng (lane 6) and 75 ng (lane 7). Amounts of PCNA used in titration were 0 ng (lane 1), 5.4 ng (lane 2), 11 ng (lane 3), 22 ng (lane 4), 43 ng (lane 5), 86 ng (lane 6) and 129 ng (lane 7). (F) Titration of PCNA in the presence of HincII. Amount of PCNA is same as (E). Reactions in (C) were carried out for 10 min under the conditions described in the Materials and Methods section. Reactions in (D–F) were carried out for 10 min under the conditions described in the Materials and Methods section except for the amount of pol δ* (140 ng). Products were analyzed by 0.7% alkaline-agarose gel electrophoresis and incorporation of dNMP were measured as described.
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Figure 6: Dynamics of replication factors in the elongation phase of DNA synthesis with pol δ*. (A) SDS–PAGE analysis of purified recombinant proteins. Pol δ* (1.9 μg) and POLD3 (0.5 μg) were loaded on a SDS 4–20% gradient polyacrylamide gel and stained with CBB. (B) Reconstitution of pol δ with POLD3 and pol δ*. Reactions were carried out for 10 min under the conditions described in the Materials and Methods section except for pol δ* (70 ng) or pol δ (90 ng) in the absence (−) or presence (+) of POLD3 (20 ng). (C–E) Titration of pol δ* (C), RFC (D) and PCNA (E). Amounts of pol δ* were 0 ng (lane 1), 4.3 ng (lane 2), 17 ng (lane 3), 35 ng (lane 4), 70 ng (lane 5), 100 ng (lane 6), and 140 ng (lane 7). Amounts of RFC used in the titration were 0 ng (lane 1), 2.3 ng (lane 2), 4.7 ng, (lane 3), 9.4 ng (lane 4), 19 ng (lane 5), 38 ng (lane 6) and 75 ng (lane 7). Amounts of PCNA used in titration were 0 ng (lane 1), 5.4 ng (lane 2), 11 ng (lane 3), 22 ng (lane 4), 43 ng (lane 5), 86 ng (lane 6) and 129 ng (lane 7). (F) Titration of PCNA in the presence of HincII. Amount of PCNA is same as (E). Reactions in (C) were carried out for 10 min under the conditions described in the Materials and Methods section. Reactions in (D–F) were carried out for 10 min under the conditions described in the Materials and Methods section except for the amount of pol δ* (140 ng). Products were analyzed by 0.7% alkaline-agarose gel electrophoresis and incorporation of dNMP were measured as described.
Mentions: The role of the 66 kDa subunit, POLD3, of pol δ in the dynamic processes involved in elongation, and the biochemical properties of subassembly (pol δ*) lacking the POLD3 subunit are of great interest. First, we examined the efficiency of DNA synthesis of human pol δ* using purified proteins (Figure 6A). A comparison of activities with equivalent amounts of pol δ* and pol δ demonstrated inefficiency of pol δ* under the standard reaction conditions with singly primed ss mp18 DNA (Figure 6B), decrease and heterogeneity in length of the products being observed with emphasized pausing sites (Figure 6B and C). The shorter products were shifted to longer ones at higher concentrations of pol δ* (Figure 6C), as with pol δ (Figure 2C). When the missing subunit, POLD3, was introduced into the reaction with pol δ*, the activity was restored to the level with pol δ (Figure 6B), indicating that the lower activity of pol δ* was due to the missing function of POLD3 subunit, rather than denaturation of proteins caused by incomplete assembly. The evidence presented here is consistent with reports for yeast counterparts (30,45) and human pol δ (36,46).Figure 6.

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