Dataset Information

Direct interaction between XRCC1 and UNG2 facilitates rapid repair of uracil in DNA by XRCC1 complexes.

ABSTRACT: Uracil-DNA glycosylase, UNG2, interacts with PCNA and initiates post-replicative base excision repair (BER) of uracil in DNA. The DNA repair protein XRCC1 also co-localizes and physically interacts with PCNA. However, little is known about whether UNG2 and XRCC1 directly interact and participate in a same complex for repair of uracil in replication foci. Here, we examine localization pattern of these proteins in live and fixed cells and show that UNG2 and XRCC1 are likely in a common complex in replication foci. Using pull-down experiments we demonstrate that UNG2 directly interacts with the nuclear localization signal-region (NLS) of XRCC1. Western blot and functional analysis of immunoprecipitates from whole cell extracts prepared from S-phase enriched cells demonstrate the presence of XRCC1 complexes that contain UNG2 in addition to separate XRCC1 and UNG2 associated complexes with distinct repair features. XRCC1 complexes performed complete repair of uracil with higher efficacy than UNG2 complexes. Based on these results, we propose a model for a functional role of XRCC1 in replication associated BER of uracil.

SUBMITTER: Akbari M

PROVIDER: S-EPMC2901844 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Direct interaction between XRCC1 and UNG2 facilitates rapid repair of uracil in DNA by XRCC1 complexes.

Akbari Mansour M Solvang-Garten Karin K Hanssen-Bauer Audun A Lieske Nora Valeska NV Pettersen Henrik Sahlin HS Pettersen Grete Klippenvåg GK Wilson David M DM Krokan Hans E HE Otterlei Marit M

DNA repair 20100513 7

Uracil-DNA glycosylase, UNG2, interacts with PCNA and initiates post-replicative base excision repair (BER) of uracil in DNA. The DNA repair protein XRCC1 also co-localizes and physically interacts with PCNA. However, little is known about whether UNG2 and XRCC1 directly interact and participate in a same complex for repair of uracil in replication foci. Here, we examine localization pattern of these proteins in live and fixed cells and show that UNG2 and XRCC1 are likely in a common complex in ...[more]

PMID: 20466601

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Project description:Two dimensional (2D) NMR and molecular dynamics simulations have been used to determine the three dimensional (3D) structure of a hairpin DNA, d-CTA-GAGGATCC-TUTT-GGATCCT (22mer; abbreviated as U2-hairpin), which has uracil at the second position from the 5' end of the tetraloop. The(1)H resonances of this hairpin have been assigned almost completely. NMR restrained molecular dynamics and energy minimization procedures have been used to describe the 3D structure of U2-hairpin. This study establishes that the stem of the hairpin adopts a right-handed B-DNA conformation, while the T(12)and T(15)nucleotides stack upon 3' and 5' ends of the stem, respectively. Further, T(14)stacks upon both T(12)and T(15). Though U(13)partially stacks upon T(14), no stacking interaction is observed between U(13)and T(12). All the individual nucleotide bases belonging to the stem and T(12)and T(15)of the loop adopt ' anti ' conformation with respect to their sugar moiety, while the U(13)and T(14)of the loop are in ' syn ' conformation. The turning phosphate in the loop is located between T(13)and T(14). This study and a concurrent NMR structural study on yet another hairpin DNA d-CTAGAGGAATAA-TTTU-GGATCCT (22mer; abbreviated as U4-hairpin), with uracil at the fourth position from the 5' end of the tetraloop throw light upon various interactions which have been reported between Escherichia coli uracil DNA glycosylase (UDG) and uracil containing DNA. The epsilon of T(12)and alpha, beta, gamma, epsilon and zeta of U(13)and gamma of T(14), which partially influence the local conformation of U(13)in U2-hairpin are all locked in ' trans ' conformation. Such stretched out backbone conformation in the vicinity of U(13)could be the reason as to why the U2-hairpin is found to be the poor substrate for its interaction with UDG compared to the other substrates in which the uracil is at first, third and fourth positions of the tetraloop from its 5' end, as reported earlier by Vinay and Varshney. This study shows that UDG actively promotes the flipping of uracil from a stacked conformation and rules out the possibility of UDG recognizing the flipped out uracil bases.

Dataset Information

Direct interaction between XRCC1 and UNG2 facilitates rapid repair of uracil in DNA by XRCC1 complexes.

Publications

Direct interaction between XRCC1 and UNG2 facilitates rapid repair of uracil in DNA by XRCC1 complexes.

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