Molecular Mechanisms of DNA Polymerase Clamp Loaders

Kelch, Brian A.; Makino, Debora L.; Simonetta, K.R.; O’Donnell, Michael; Kuriyan, John

doi:10.1007/978-94-007-2530-0_10

Cited by 2 publications

(7 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The upper tier is a sealed ring consisting of five collar domains - one from each subunit, whereas the lower tier is composed of five AAA+ modules and is an open spiral with a gate between the first and the fifth subunits. However, the first subunit of the eukaryotic and T4 phage clamp loaders contain an additional A’ domain at their respective C termini that contacts the fifth subunit and closes the spiral gap ( Kelch et al, 2012b ). The five ATP binding sites are located at the interfaces between adjacent subunits, with one subunit contributing the Walker A (P-loop) and Walker B (DExx box, where the x can be any residue) motifs, and the other contributing the SRC motif that harbors an essential ‘arginine finger’ residue ( Johnson et al, 2006 ).…”

Section: Introductionmentioning

confidence: 99%

“…All studies thus far have revealed that primer/template (P/T) DNA with a 3′-recessed end binds inside the chamber of the ATP-bound clamp loader ( Figure 1b ; Bowman et al, 2005 ; Kelch et al, 2011 ). Moreover, clamp loaders of bacterial and eukaryotic systems only need ATP binding to crack open the clamp ring; neither ATP hydrolysis nor DNA is required ( Hingorani and O’Donnell, 1998 ; Kelch et al, 2012b ; Kelch et al, 2012a ; Turner et al, 1999 ; Zhuang et al, 2006 ). DNA binding appears to properly engage the ATPase sites for hydrolysis, leading to closure of the clamp around the duplex and dissociation of the clamp loader from DNA ( Anderson et al, 2009 ; Chen et al, 2009 ; Gaubitz et al, 2022 ; Kelch et al, 2012b ; Liu et al, 2017 ; Marzahn et al, 2015 ; Sakato et al, 2012a ; Trakselis et al, 2003 ).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cryo-EM structures reveal that RFC recognizes both the 3′- and 5′-DNA ends to load PCNA onto gaps for DNA repair

Zheng

Georgescu

Yao

et al. 2022

eLife

View full text Add to dashboard Cite

RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases δ and ε. The RFC pentamer forms a central chamber that binds 3′ ss/ds DNA junctions to load PCNA onto DNA during replication. We show here five structures that identify a second DNA binding site in RFC that binds a 5′ duplex. This 5′ DNA site is located between the N-terminal BRCT domain and AAA+ module of the large Rfc1 subunit. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nt gaps, consistent with the structural results. Because both 3′ and 5′ ends are present at a ssDNA gap, we propose that the 5′ site facilitates RFC’s PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5′ DNA binding domain of Rfc1. We further observe that a 5′ end facilitates PCNA loading at an RPA coated 30-nt gap, suggesting a potential role of the RFC 5′-DNA site in lagging strand DNA synthesis.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Cryo-EM structures reveal that RFC recognizes both the 3′- and 5′-DNA ends to load PCNA onto gaps for DNA repair

Zheng

Georgescu

Yao

et al. 2022

eLife

View full text Add to dashboard Cite

show abstract

“…Biochemical studies have shown that ATP binding enables clamp loader-clamp binding and opening in both eukaryotes and bacterial systems, illustrated in the first two diagrams of Fig. 6 (Hingorani and O’Donnell, 1998; Kelch et al, 2012a; Kelch et al, 2012b; Turner et al, 1999; Zhuang et al, 2006). Recent structural studies of RFC-PCNA-ATPγS reveal that the RFC-PCNA open form is sufficiently wide to admit dsDNA into the 3’-DNA1 site ( top path in Fig.…”

Section: Discussionmentioning

confidence: 99%

“…1b ) (Bowman et al, 2005; Kelch et al, 2011). Moreover, clamp loaders of bacterial and eukaryotic systems only need ATP binding to crack open the clamp ring; neither ATP hydrolysis nor DNA is required (Kelch et al, 2012a,b; Turner et al, 1999). DNA binding appears to properly engage the ATPase sites for hydrolysis, leading to RFC dissociation from DNA and PCNA closure around the duplex (Gaubitz et al, 2021; Kelch et al, 2012a).…”

Section: Introductionmentioning

confidence: 99%

“…1b ) (Bowman et al, 2005; Kelch et al, 2011). Moreover, clamp loaders of bacterial and eukaryotic systems only need ATP binding to crack open the clamp ring; neither ATP hydrolysis nor DNA is required (Hingorani and O’Donnell, 1998; Kelch et al, 2012a; Kelch et al, 2012b; Turner et al, 1999; Zhuang et al, 2006). DNA binding appears to properly engage the ATPase sites for hydrolysis, leading to closure of the clamp around the duplex and dissociation of the clamp loader from DNA (Anderson et al, 2009; Chen et al, 2009; Gaubitz et al, 2022; Kelch et al, 2012a; Liu et al, 2017; Marzahn et al, 2015; Sakato et al, 2012a; Trakselis et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cryo-EM structures reveal that RFC recognizes both the 3’- and 5’-DNA ends to load PCNA onto gaps for DNA repair

Zhang

Georgescue

Yao

et al. 2022

Preprint

View full text Add to dashboard Cite

RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases delta and epsilon;. The RFC pentamer forms a central chamber that binds a 3 primed DNA junction to load PCNA onto DNA during replication. We show here five structures revealing a novel second DNA binding site in RFC that binds a 5 prime junction. This 5 prime DNA site is located in the N-terminal BRCT domain and AAA module of the large Rfc1 subunit. It appears that 5 prime DNA binds after 3 prime DNA binding to RFC. Our structures reveal ideal binding to a 7-nt gap, which includes 2 bp unwound by the clamp loader. Biochemical studies show enhanced binding to 5 and 10 nucleotide gaps, consistent with the structural results. We propose that the 5 prime DNA site facilitates RFC PCNA loading activity at a DNA damage-induced gap to recruit gap-filling polymerases. These findings are consistent with genetic studies showing that base excision repair of gaps greater than 1 base requires PCNA and involves the 5 prime DNA binding domain of Rfc1. We further observe that a 5 prime site facilitates PCNA loading at an RPA coated 50-nt gap, suggesting a potential role of the RFC 5 prime DNA site at longer gaps including during lagging strand DNA synthesis.

show abstract

Molecular Mechanisms of DNA Polymerase Clamp Loaders

Cited by 2 publications

References 56 publications

Cryo-EM structures reveal that RFC recognizes both the 3′- and 5′-DNA ends to load PCNA onto gaps for DNA repair

Cryo-EM structures reveal that RFC recognizes both the 3′- and 5′-DNA ends to load PCNA onto gaps for DNA repair

Cryo-EM structures reveal that RFC recognizes both the 3’- and 5’-DNA ends to load PCNA onto gaps for DNA repair

Contact Info

Product

Resources

About