Results tagged “DNA Repair” from The SIBYLS Beamline

Tsutakawa et al. have combined solution Small Angle X-ray Scattering (SAXS) data collected at the SIBYLS beamline with computational modeling carried out at Oak Ridge Leadership Computing Facility ( OLCF ) to elucidate new modes of flexibility in a key protein complex (Ubiquitin-PCNA) involved in DNA replication and repair. The work was published in the Oct 25th issue of PNAS, and has been highlighted by OLCF and the Faculty of 1000.

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PCNA ubiquitination in response to DNA damage leads to the recruitment of specialized translesion polymerases to the damage locus. This constitutes one of the initial steps in translesion synthesis (TLS)-a critical pathway for cell survival and for maintenance of genome stability. The recent crystal structure of ubiquitinated PCNA (Ub-PCNA) sheds light on the mode of association between the two proteins but also revealed that paradoxically, the ubiquitin surface engaged in PCNA interactions was the same as the surface implicated in translesion polymerase binding. This finding implied a degree of flexibility inherent in the Ub-PCNA complex that would allow it to transition into a conformation competent to bind the TLS polymerase. To address the issue of segmental flexibility, we combined multiscale computational modeling and small angle X-ray scattering. This combined strategy revealed alternative positions for ubiquitin to reside on the surface of the PCNA homotrimer, distinct from the position identified in the crystal structure. Two mutations originally identified in genetic screens and known to interfere with TLS are positioned directly beneath the bound ubiquitin in the alternative models. These computationally derived positions, in an ensemble with the crystallographic and flexible positions, provided the best fit to the solution scattering, indicating that ubiquitin dynamically associated with PCNA and is capable of transitioning between a few discrete sites on the PCNA surface. The finding of new docking sites and the positional equilibrium of PCNA-Ub occurring in solution provide unexpected insight into previously unexplained biological observations

Tsutakawa SE, Van Wynsberghe AW, Freudenthal BD, Weinacht CP, Gakhar L, Washington MT, Zhuang Z, Tainer JA, Ivanov I. “Solution X-ray scattering combined with computational modeling reveals multiple conformations of covalently bound ubiquitin on PCNA.” Proc Natl Acad Sci U S A. 2011 Oct 17.

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Two related manuscripts appear in the April 15 issue of CELL reporting results endo and exo nucleases important for DNA repair. Both groups of researchers made extensive use of the MX capabilities of the SIBYLS beamline to collect critical crystallographic data. The crystal structures of human flap endonuclease (FEN1) in complex with both substrate and product forms of DNA were reported by Susan Tsutakawa et al. in the following manuscript:

Tsutakawa SE, Classen S, Chapados BR, Arvai A, Finger DL, Guenther G, Tomlinson CG, Thompson P, Sarker AH, Shen B, Cooper PK, Grasby JA, and Tainer JA. “ Human Flap Endonuclease Structures, DNA Double Base Flipping and a Unified Understanding of the FEN1 Superfamily. ” Cell, Volume 145, Issue 2, 198-211, 15 April 2011

Published simultaneously by Jill Orans et al is the crystal structure of human exonuclease 1 in complex with DNA substrate.

Orans J, McSweeney EA, Iyer RR, Hast MA, Hellinga HW, Modrich P, Beese LS. “ Structures of Human Exonuclease 1 DNA Complexes Suggest a Unified Mechanism for Nuclease Family. ” Cell, Volume 145, Issue 2, 212-223, 15 April 2011

Together these two manuscripts present exciting insights into the molecular principles governing diverse endo- and exonucleolytic cleavage specificities of members of the RAD2/FEN superfamily of nucleases, which have critical roles in DNA replication and maintenance.

A paper has been published in The October 2 issue of Cell by Scott Williams et al. that sheds light on a previously missing piece of the double-strand break repair complex MRN (aka Mre11-Rad50-Nbs1). The paper entitled “Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair” presents a compelling model of the role of Nbs1 (i.e “N” of the MRN) in coordinating double-strand break processing and repair. The paper was made possible in part by crystal structures and SAXS data of Nbs1 that were collected at the SIBYLS beamline.

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Abstract:

The Nijmegen breakage syndrome 1 (Nbs1) subunit of the Mre11-Rad50-Nbs1 (MRN) complex protects genome integrity by coordinating double-strand break (DSB) repair and checkpoint signaling through undefined interactions with ATM, MDC1, and Sae2/Ctp1/CtIP. Here, fission yeast and human Nbs1 structures defined by X-ray crystallography and small angle X-ray scattering (SAXS) reveal Nbs1 cardinal features: fused, extended, FHA-BRCT1-BRCT2 domains flexibly linked to C-terminal Mre11- and ATM-binding motifs. Genetic, biochemical, and structural analyses of an Nbs1-Ctp1 complex show Nbs1 recruits phosphorylated Ctp1 to DSBs via binding of the Nbs1 FHA domain to a Ctp1 pThr-Asp motif. Nbs1 structures further identify an extensive FHA-BRCT interface, a divalent MDC1-binding scaffold, an extended conformational switch, and the molecular consequences associated with cancer predisposing Nijmegen breakage syndrome mutations. Tethering of Ctp1 to a flexible Nbs1 arm suggests a mechanism for restricting DNA end processing and homologous recombination activities of Sae2/Ctp1/CtIP to the immediate vicinity of DSBs.

Williams RS, Dodson GE, Limbo O, Yamada Y, Williams JS, Guenther G, Classen S, Glover MJN, Iwasaki H, Russell P, Tainer JA. “Nbs1 Flexibly Tethers Ctp1 and Mre11-Rad50 to Coordinate DNA Double-Strand Break Processing and Repair” Cell, Volume 139, Issue 1, 87-99, 2 October 2009
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In this paper, published in Nucleic Acid Research, the authors used small-angle X-ray scattering (SAXS) combined with advanced computational approaches to characterize the conformational variability and DNA-binding properties of PNK. Extensive use of the SAXS capabilities of the SIBYLS beamline allowed the authors to visualize a flexible attachment of the FHA domain to the catalytic segment and localize the DNA in the DNA/PNK complex.

Bernstein NK, Hammel M, Mani RS, Weinfeld M, Pelikan M, Tainer JA, Glover JN. "Mechanism of DNA substrate recognition by the mammalian DNA repair enzyme, Polynucleotide Kinase." Nucleic Acids Res. 2009 Aug 11. [epub].link out



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An excellent paper came out today in the Oct 3rd issue of Cell detailing structural, biochemical, and genetic studies of the Mre11-DNA complex and its role in detecting and repairing double-strand breaks in DNA. Both the SAXS and crystallography data were collected at the SIBYLS beamline. There is also a nice writeup by Paul Preuss which appears in the todays Berkeley Labs News Release.

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Mre11 dimers coordinate DNA end-bridging and nuclease processing in double-strand break repair” by R. Scott Williams, Gabriel Moncalian, Jessica S. Williams, Yoshiki Yamada, Oliver Limbo, David S. Shin, Lynda M. Groocock, Dana Cahill, Chiharu Hitomi, Grant Guenther, Davide Moiani, James P. Carney, Paul Russell, and John A. Tainer, appears in the 3 October 2008 issue of Cell.

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