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Updated: Jul 24, 2025

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay
Published on: February 3, 2022
Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification on biology
Fengwei Zheng1, Roxana E Georgescu2, Nina Y Yao2
1Department of Structural Biology, Van Andel Institute, Grand Rapids, MI, USA.
Abstract:
Rad24-RFC (replication factor C) loads the 9-1-1 checkpoint clamp onto the recessed 5' ends by binding a 5' DNA at an external surface site and threading the 3' single-stranded DNA (ssDNA) into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' end, thus presumably leaving 9-1-1 on duplex 3' ss/double-stranded DNA (dsDNA) after Rad24-RFC ejects from DNA. We captured five Rad24-RFC-9-1-1 loading intermediates using a 10-nt gap DNA. We also determined the structure of Rad24-RFC-9-1-1 using a 5-nt gap DNA. The structures reveal that Rad24-RFC is unable to melt DNA ends and that a Rad24 loop limits the dsDNA length in the chamber. These observations explain Rad24-RFC's preference for a preexisting gap of over 5-nt ssDNA and suggest a direct role of the 9-1-1 in gap repair with various TLS (trans-lesion synthesis) polymerases in addition to signaling the ATR kinase.
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