Flexibility and Mechanism of Ub-PCNA in DNA Repair

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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.

ub-pcna-brownian.tif

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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