Limits...
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.

Show MeSH

Related in: MedlinePlus

A model for dynamics of replication factors during pol δ dissociation–association cycles. The elongation complex consists of RFC, PCNA and pol δ in the elongation phase of DNA replication (Stage I). Pol δ contacts with PCNA and prevents RFC from dissociating. Contribution of RFC–RPA interaction for stable association in the complex has been proposed (26). Pathway 1, dissociation of pol δ leaving RFC and PCNA on DNA (Stage II). DNA–RFC–PCNA complex formation could be coupled with dissociation of pol δ, mediated by the POLD3 subunit. Pathway 2, reassociation of pol δ to form the elongation complex. The POLD3 subunit of pol δ might mediate efficient transfer of PCNA from RFC to pol δ. Pathway 3, unloading or sliding of PCNA out of the primer terminus, leaving RFC (Stage III). RFC probably interacts with RPA for retaining around primer terminus (26). Pathway 4, reloading of PCNA from solution or PCNA sliding back along the DNA to reform the DNA–RFC–PCNA complex. Pathway 5, dissociation of RFC from DNA (Stage IV). The main pathways are shown as thick arrows.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: A model for dynamics of replication factors during pol δ dissociation–association cycles. The elongation complex consists of RFC, PCNA and pol δ in the elongation phase of DNA replication (Stage I). Pol δ contacts with PCNA and prevents RFC from dissociating. Contribution of RFC–RPA interaction for stable association in the complex has been proposed (26). Pathway 1, dissociation of pol δ leaving RFC and PCNA on DNA (Stage II). DNA–RFC–PCNA complex formation could be coupled with dissociation of pol δ, mediated by the POLD3 subunit. Pathway 2, reassociation of pol δ to form the elongation complex. The POLD3 subunit of pol δ might mediate efficient transfer of PCNA from RFC to pol δ. Pathway 3, unloading or sliding of PCNA out of the primer terminus, leaving RFC (Stage III). RFC probably interacts with RPA for retaining around primer terminus (26). Pathway 4, reloading of PCNA from solution or PCNA sliding back along the DNA to reform the DNA–RFC–PCNA complex. Pathway 5, dissociation of RFC from DNA (Stage IV). The main pathways are shown as thick arrows.

Mentions: We here propose a model for dynamics of replication factors during a dissociation–association cycle of pol δ (Figure 8). The elongation complex consists of RFC, PCNA and pol δ (Figure 8, stage I). Pol δ dissociates frequently from growing 3′-hydroxyl ends and PCNA during elongation of DNA replication (Figure 8, pathway 1). Then, PCNA from which pol δ has detached would be held by the remaining RFC (Figure 8, stage II). If pol δ reassociated quickly (Figure 8, pathway 2), RFC could remain around the primer terminus, and travel with pol δ and PCNA during elongation of DNA replication. In this cycle, PCNA is not released out of the replication complex, which implies ‘recycling PCNA’ (Figure 8). Since the DNA–RFC–PCNA complex (stage II) is not stable (52), the RFC could unload PCNA, or release the PCNA out of the primer terminus (Figure 8, pathway 3). Probably, the unloading reaction does not predominate, as shown in yeast RFC (53). If PCNA were available from solution or along the DNA, RFC could incorporate PCNA into the complex (Figure 8, pathway 4). Otherwise, RFC would dissociate from the primer terminus (Figure 8, pathway 5). At higher concentrations greater than saturated amounts of RFC, the size of products was increased (Figures 2D and F, 3D and F). Probably, with such concentrations, RFC is sufficient for initiation and remaining RFC in solution could help to overcome the pausing site, implying dissociation of RFC at strong pausing sites. However, this was negligible under normal replication conditions, and was detectable in reactions omitting pol δ, as shown in lane 6 of Figure 5B. This suggested that pol δ prevents dissociation of RFC. In intensive studies of PCNA-loading reactions with yeast RFC, no stable RFC–DNA complex was detected in the absence of PCNA and only became detectable in the presence of ATPγS, and RFC dissociated quickly from DNA after loading PCNA (47,48). Therefore, it has been considered that RFC is absent in elongation complexes. Our results may explain the discrepancy regarding the prevention of dissociation of RFC by sequential loading of PCNA (pathway 4) and pol δ (pathway 2) in the replication assay, but not PCNA loading assays. Therefore, we consider that our model is consistent with the previous observations (38,47,48). Gomes et al. (47) has also proposed a loading pathway, in which PCNA–RFC complex first forms an ATP-dependent ring-opened complex and subsequently associates with DNA and delivers PCNA to the template–primer junction. RFC associated with the DNA cannot recruit PCNA nor load it at termini, first having to dissociate coupled with ATP hydrolysis (47). Our model is consistent with the loading mechanism. RFC is probably detached from the primer terminus before loading PCNA, but associated around primer terminus via interaction with RPA (stage III) as proposed previously (26).Figure 8.


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)

A model for dynamics of replication factors during pol δ dissociation–association cycles. The elongation complex consists of RFC, PCNA and pol δ in the elongation phase of DNA replication (Stage I). Pol δ contacts with PCNA and prevents RFC from dissociating. Contribution of RFC–RPA interaction for stable association in the complex has been proposed (26). Pathway 1, dissociation of pol δ leaving RFC and PCNA on DNA (Stage II). DNA–RFC–PCNA complex formation could be coupled with dissociation of pol δ, mediated by the POLD3 subunit. Pathway 2, reassociation of pol δ to form the elongation complex. The POLD3 subunit of pol δ might mediate efficient transfer of PCNA from RFC to pol δ. Pathway 3, unloading or sliding of PCNA out of the primer terminus, leaving RFC (Stage III). RFC probably interacts with RPA for retaining around primer terminus (26). Pathway 4, reloading of PCNA from solution or PCNA sliding back along the DNA to reform the DNA–RFC–PCNA complex. Pathway 5, dissociation of RFC from DNA (Stage IV). The main pathways are shown as thick arrows.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: A model for dynamics of replication factors during pol δ dissociation–association cycles. The elongation complex consists of RFC, PCNA and pol δ in the elongation phase of DNA replication (Stage I). Pol δ contacts with PCNA and prevents RFC from dissociating. Contribution of RFC–RPA interaction for stable association in the complex has been proposed (26). Pathway 1, dissociation of pol δ leaving RFC and PCNA on DNA (Stage II). DNA–RFC–PCNA complex formation could be coupled with dissociation of pol δ, mediated by the POLD3 subunit. Pathway 2, reassociation of pol δ to form the elongation complex. The POLD3 subunit of pol δ might mediate efficient transfer of PCNA from RFC to pol δ. Pathway 3, unloading or sliding of PCNA out of the primer terminus, leaving RFC (Stage III). RFC probably interacts with RPA for retaining around primer terminus (26). Pathway 4, reloading of PCNA from solution or PCNA sliding back along the DNA to reform the DNA–RFC–PCNA complex. Pathway 5, dissociation of RFC from DNA (Stage IV). The main pathways are shown as thick arrows.
Mentions: We here propose a model for dynamics of replication factors during a dissociation–association cycle of pol δ (Figure 8). The elongation complex consists of RFC, PCNA and pol δ (Figure 8, stage I). Pol δ dissociates frequently from growing 3′-hydroxyl ends and PCNA during elongation of DNA replication (Figure 8, pathway 1). Then, PCNA from which pol δ has detached would be held by the remaining RFC (Figure 8, stage II). If pol δ reassociated quickly (Figure 8, pathway 2), RFC could remain around the primer terminus, and travel with pol δ and PCNA during elongation of DNA replication. In this cycle, PCNA is not released out of the replication complex, which implies ‘recycling PCNA’ (Figure 8). Since the DNA–RFC–PCNA complex (stage II) is not stable (52), the RFC could unload PCNA, or release the PCNA out of the primer terminus (Figure 8, pathway 3). Probably, the unloading reaction does not predominate, as shown in yeast RFC (53). If PCNA were available from solution or along the DNA, RFC could incorporate PCNA into the complex (Figure 8, pathway 4). Otherwise, RFC would dissociate from the primer terminus (Figure 8, pathway 5). At higher concentrations greater than saturated amounts of RFC, the size of products was increased (Figures 2D and F, 3D and F). Probably, with such concentrations, RFC is sufficient for initiation and remaining RFC in solution could help to overcome the pausing site, implying dissociation of RFC at strong pausing sites. However, this was negligible under normal replication conditions, and was detectable in reactions omitting pol δ, as shown in lane 6 of Figure 5B. This suggested that pol δ prevents dissociation of RFC. In intensive studies of PCNA-loading reactions with yeast RFC, no stable RFC–DNA complex was detected in the absence of PCNA and only became detectable in the presence of ATPγS, and RFC dissociated quickly from DNA after loading PCNA (47,48). Therefore, it has been considered that RFC is absent in elongation complexes. Our results may explain the discrepancy regarding the prevention of dissociation of RFC by sequential loading of PCNA (pathway 4) and pol δ (pathway 2) in the replication assay, but not PCNA loading assays. Therefore, we consider that our model is consistent with the previous observations (38,47,48). Gomes et al. (47) has also proposed a loading pathway, in which PCNA–RFC complex first forms an ATP-dependent ring-opened complex and subsequently associates with DNA and delivers PCNA to the template–primer junction. RFC associated with the DNA cannot recruit PCNA nor load it at termini, first having to dissociate coupled with ATP hydrolysis (47). Our model is consistent with the loading mechanism. RFC is probably detached from the primer terminus before loading PCNA, but associated around primer terminus via interaction with RPA (stage III) as proposed previously (26).Figure 8.

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