Biology subjects

Yao, N. Y.

Publications and source records attributed to Yao, N. Y..

6 recordsLinked to original sources

bioRxiv2026

The E. coli clamp loader sharply bends DNA to load β-clamp onto small gaps

DNA sliding clamps are essential for processive DNA synthesis in all domains of life and are loaded by ATP-dependent clamp loaders that recognize recessed 3' ends. How clamp loaders function at nicks and small ssDNA gaps--common intermediates during DNA repair--remains incompletely understood. Here, we show that the bacterial Escherichia coli DnaX clamp loader employs a fundamentally different mechanism from its eukaryotic counterpart. Whereas eukaryotic RFC unwinds DNA at the recessed 3' end and stabilizes the 5'-dsDNA at a dedicated shoulder site, the bacterial DnaX-complex neither unwinds DNA nor stably binds the 5'-dsDNA in vitro. Instead, cryo-EM structures reveal that the {beta}-clamp itself contains a conserved external DNA-binding site that enables sharp bending of gapped DNA by [~]150{degrees}, promoting insertion of the 3'-dsDNA into the clamp. This DNA-bending mechanism allows efficient {beta}-clamp loading at nicks and small gaps and reveals a distinct bacterial strategy for clamp loading. Because small DNA gaps are frequently associated with DNA damage, clamps loaded at these sites are likely important for DNA repair. In briefZheng et al. show that the bacterial clamp loader DnaX-complex uses a DNA-bending mechanism--rather than DNA unwinding--to load the {beta}-clamp at nicks and small gaps, revealing a clamp-loading strategy distinct from eukaryotic RFC and relevant to DNA damage repair. HighlightsO_LIThe bacterial DnaX clamp loader lacks a stable shoulder DNA-binding site C_LIO_LIUnlike eukaryotic RFC, DnaX does not unwind DNA at nicks and small gaps C_LIO_LIThe E. coli {beta}-clamp contains a conserved external DNA-binding site absent in PCNA C_LIO_LISharp DNA bending enables {beta}-clamp loading at nicks and small ssDNA gaps C_LI

biochemistry↗

bioRxiv2023

Structures of 9-1-1 DNA checkpoint clamp loading at gaps from start to finish and ramification to biology

Recent structural studies show the Rad24-RFC loads the 9-1-1 checkpoint clamp onto a recessed 5' end by binding the 5' DNA on Rad24 at an external surface site and threading the 3' ssDNA into the well-established internal chamber and into 9-1-1. We find here that Rad24-RFC loads 9-1-1 onto DNA gaps in preference to a recessed 5' DNA end, thus presumably leaving 9-1-1 on a 3' ss/ds DNA after Rad24-RFC ejects from the 5' gap end and may explain reports of 9-1-1 directly functioning in DNA repair with various TLS polymerases, in addition to signaling the ATR kinase. To gain a deeper understanding of 9-1-1 loading at gaps we report high-resolution structures of Rad24-RFC during loading of 9-1-1 onto 10-nt and 5-nt gapped DNAs. At a 10-nt gap we captured five Rad24-RFC-9-1-1 loading intermediates in which the 9-1-1 DNA entry gate varies from fully open to fully closed around DNA using ATP{gamma}S, supporting the emerging view that ATP hydrolysis is not needed for clamp opening/closing, but instead for dissociation of the loader from the clamp encircling DNA. The structure of Rad24-RFC-9-1-1 at a 5-nt gap shows a 180{degrees} axially rotated 3'-dsDNA which orients the template strand to bridge the 3'- and 5'- junctions with a minimum 5-nt ssDNA. The structures reveal a unique loop on Rad24 that limits the length of dsDNA in the inner chamber, and inability to melt DNA ends unlike RFC, thereby explaining Rad24-RFCs preference for a preexisting ssDNA gap and suggesting a direct role in gap repair in addition to its checkpoint role.

biochemistry↗

bioRxiv2022

A novel 5' DNA binding site in RFC facilitates PCNA loading for gap DNA repair

RFC uses ATP to assemble PCNA onto primed sites for replicative DNA polymerases {delta} and {varepsilon}. 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 2nd 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 RFCs 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

biochemistry↗

bioRxiv2021

Rad24-RFC loads the 9-1-1 clamp by inserting DNA from the top of a wide-open ring, opposite the mechanism of RFC/PCNA

In response to DNA damage, the ring-shaped 9-1-1 clamp is loaded onto 5 recessed DNA to arrest the cell cycle and activate the DNA damage checkpoint. The 9-1-1 clamp is a heterotrimeric ring that is loaded in S. cerevisiae by Rad24-RFC, an alternative clamp loader in which Rad24 replaces the Rfc1 subunit in the RFC1-5 clamp loader of PCNA. Unlike RFC that loads the PCNA ring onto a 3-ss/ds DNA junction, Rad24-RFC loads the 9-1-1 ring onto a 5-ss/ds DNA junction, a consequence of DNA damage. The underlying 9-1-1 clamp loading mechanism has been a mystery. Here we report two 3.2-[A] cryo-EM structures of Rad24-RFC bound to DNA and either a closed or 27 [A] open 9-1-1 clamp. The structures reveal a completely unexpected mechanism by which a clamp can be loaded onto DNA. The Rad24 subunit specifically recognizes the 5-DNA junction and holds ds DNA outside the clamp loader and above the plane of the 9-1-1 ring, rather than holding DNA inside and below the clamp as in RFC. The 3 ssDNA overhang is required to obtain the structure, and thus confers a second DNA binding site. The bipartite DNA binding by Rad24-RFC suggests that ssDNA may be flipped into the open 9-1-1 ring, similar to ORC-Cdc6 that loads the Mcm2-7 ring on DNA. We propose that entry of ssDNA through the 9-1-1 ring triggers the ATP hydrolysis and release of the Rad24-RFC. The key DNA binding residues are conserved in higher eukaryotes, and thus the 9-1-1 clamp loading mechanism likely generalizes.

biochemistry↗

bioRxiv2019

Ctf4 organizes sister replisomes and Pol α into a replication factory

The current view is that eukaryotic replisomes are independent. Here we show that Ctf4 tightly dimerizes CMG helicase, with an extensive interface involving Psf2, Cdc45, and Sld5. Interestingly, Ctf4 binds only one Pol -primase. Thus, Ctf4 may have evolved as a trimer to organize two helicases and one Pol -primase into a replication factory. In the 2CMG-Ctf43-1Pol -primase factory model, the two CMGs nearly face each other, placing the two lagging strands toward the center and two leading strands out the sides. The single Pol -primase is centrally located and may prime both sister replisomes. The Ctf4-coupled-sister replisome model is consistent with cellular microscopy studies revealing two sister forks of an origin remain attached and are pushed forward from a protein platform. The replication factory model may facilitate parental nucleosome transfer during replication.

biochemistry↗

bioRxiv2017

Mcm10 functions to isomerize CMG-DNA for replisome bypass of DNA blocks

Replicative helicases of all cell types are rings that unwind DNA by steric exclusion in which the helicase ring only encircles the tracking strand, excluding the other strand outside the ring. Steric exclusion mediated unwinding enables helicase rings to bypass blocks on the strand that is excluded from the central channel. Unlike other replicative helicases, eukaryotic CMG encircles duplex DNA at a forked junction and is stopped by a block on the non-tracking (lagging) strand. This report demonstrates that Mcm10, an essential replication protein unique to eukaryotes, binds CMG and enables the replisome to bypass blocks on the non-tracking strand, implying that Mcm10 isomerizes the CMG-DNA complex to position only one strand through the central channel. A similar CMG-DNA isomerization is needed at the origin for head-to-head CMGs to bypass one another during formation of bidirectional replication forks.

biochemistry↗