Unexpected new insights into DNA clamp loaders

Abstract

Clamp loaders are pentameric AAA+ assemblies that use ATP to open and close circular DNA sliding clamps around DNA. Clamp loaders show homology in all organisms, from bacteria to human. The eukaryotic PCNA clamp is loaded onto 3′ primed DNA by the replication factor C (RFC) hetero‐pentameric clamp loader. Eukaryotes also have three alternative RFC‐like clamp loaders (RLCs) in which the Rfc1 subunit is substituted by another protein. One of these is the yeast Rad24‐RFC (Rad17‐RFC in human) that loads a 9‐1‐1 heterotrimer clamp onto a recessed 5′ end of DNA. Recent structural studies of Rad24‐RFC have discovered an unexpected 5′ DNA binding site on the outside of the clamp loader and reveal how a 5′ end can be utilized for loading the 9‐1‐1 clamp onto DNA. In light of these results, new studies reveal that RFC also contains a 5′ DNA binding site, which functions in gap repair. These studies also reveal many new features of clamp loaders. As reviewed herein, these recent studies together have transformed our view of the clamp loader mechanism.

Structure of the PCNA unloader Elg1-RFC

“…S5), making it incompatible with DNA binding. The Elg1 plug is distinct from and is in addition to the well-established Rfc5 plug that interacts with the major groove of the dsDNA inside the central chamber of a clamp loader ( 49 ). DNA binding inside the central chamber, formed by all five subunits and accessed by widening the A-gate between the A and A′ domains of Rfc1 (or Rad24-RFC), is a required feature of both RFC (the PCNA loader) and Rad24-RFC (the 9-1-1 loader).…”

“…Previous work showed that RFC encircles the ss/dsDNA junction in the inner chamber of the clamp loader during PCNA loading ( 6 , 49 , 51 ). RFC binding to 3′ ss/dsDNA requires a sharp bend in the DNA, thought to specify loading to a nick or gap and not enabling loading on closed dsDNA ( 52 ).…”

“…Together, these unique features suggest that ATP may not be hydrolyzed in the Elg1 ATP binding site. Both Elg1 and RFC can unload PCNA from a sealed DNA Previous work showed that RFC encircles the ss/dsDNA junction in the inner chamber of the clamp loader during PCNA loading (6,49,51). RFC binding to 3′ ss/dsDNA requires a sharp bend in the DNA, thought to specify loading to a nick or gap and not enabling loading on closed dsDNA (52).…”

Differences between bacteria and eukaryotes in clamp loader mechanism, a conserved process underlying DNA replication

“…Moreover, our previous fluorescence data indicate that the E. coli clamp loader is unable to unwind a DNA p/t-junction ( 27 ). RFC’s unwinding activity is thought to facilitate its role in loading at nicked DNA for performing long-patch Base Excision Repair ( 29 , 30 , 62 ). Because the E. coli clamp loader is not known to play a role in Base Excision Repair, presumably it does not require DNA unwinding activity.…”

Differences in clamp loader mechanism between bacteria and eukaryotes

“…Moreover, our previous fluorescence data indicate that the E. coli clamp loader is unable to unwind a DNA p/t-junction 27 . RFC’s unwinding activity is thought to facilitate its role in loading at nicked DNA for performing long-patch Base Excision Repair 29,30,61 . Because the E. coli clamp loader is not known to play a role in Base Excision Repair, presumably it does not require DNA unwinding activity.…”