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The unstructured C-terminal extension of UvrD interacts with UvrB, but is dispensable for nucleotide excision repair.

ABSTRACT: During nucleotide excision repair (NER) in bacteria the UvrC nuclease and the short oligonucleotide that contains the DNA lesion are removed from the post-incision complex by UvrD, a superfamily 1A helicase. Helicases are frequently regulated by interactions with partner proteins, and immunoprecipitation experiments have previously indicated that UvrD interacts with UvrB, a component of the post-incision complex. We examined this interaction using 2-hybrid analysis and surface plasmon resonance spectroscopy, and found that the N-terminal domain and the unstructured region at the C-terminus of UvrD interact with UvrB. We analysed the properties of a truncated UvrD protein that lacked the unstructured C-terminal region and found that it showed a diminished affinity for single-stranded DNA, but retained the ability to displace both UvrC and the lesion-containing oligonucleotide from a post-incision nucleotide excision repair complex. The interaction of the C-terminal region of UvrD with UvrB is therefore not an essential feature of the mechanism by which UvrD disassembles the post-incision complex during NER. In further experiments we showed that PcrA helicase from Bacillus stearothermophilus can also displace UvrC and the excised oligonucleotide from a post-incision NER complex, which supports the idea that PcrA performs a UvrD-like function during NER in gram-positive organisms.

SUBMITTER: Manelyte L

PROVIDER: S-EPMC2997466 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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The unstructured C-terminal extension of UvrD interacts with UvrB, but is dispensable for nucleotide excision repair.

Manelyte Laura L Guy Colin P CP Smith Rachel M RM Dillingham Mark S MS McGlynn Peter P Savery Nigel J NJ

DNA repair 20090916 11

During nucleotide excision repair (NER) in bacteria the UvrC nuclease and the short oligonucleotide that contains the DNA lesion are removed from the post-incision complex by UvrD, a superfamily 1A helicase. Helicases are frequently regulated by interactions with partner proteins, and immunoprecipitation experiments have previously indicated that UvrD interacts with UvrB, a component of the post-incision complex. We examined this interaction using 2-hybrid analysis and surface plasmon resonance ...[more]

PMID: 19762288

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Project description:The incorporation of ribonucleotides in DNA has attracted considerable notice in recent years, since the pool of ribonucleotides can exceed that of the deoxyribonucleotides by at least 10-20-fold, and single ribonucleotide incorporation by DNA polymerases appears to be a common event. Moreover ribonucleotides are potentially mutagenic and lead to genome instability. As a consequence, errantly incorporated ribonucleotides are rapidly repaired in a process dependent upon RNase H enzymes. On the other hand, global genomic nucleotide excision repair (NER) in prokaryotes and eukaryotes removes damage caused by covalent modifications that typically distort and destabilize DNA through the production of lesions derived from bulky chemical carcinogens, such as polycyclic aromatic hydrocarbon metabolites, or via crosslinking. However, a recent study challenges this lesion-recognition paradigm. The work of Vaisman et al. (2013) [34] reveals that even a single ribonucleotide embedded in a deoxyribonucleotide duplex is recognized by the bacterial NER machinery in vitro. In their report, the authors show that spontaneous mutagenesis promoted by a steric-gate pol V mutant increases in uvrA, uvrB, or uvrC strains lacking rnhB (encoding RNase HII) and to a greater extent in an NER-deficient strain lacking both RNase HI and RNase HII. Using purified UvrA, UvrB, and UvrC proteins in in vitro assays they show that despite causing little distortion, a single ribonucleotide embedded in a DNA duplex is recognized and doubly-incised by the NER complex. We present the hypothesis to explain the recognition and/or verification of this small lesion, that the critical 2'-OH of the ribonucleotide - with its unique electrostatic and hydrogen bonding properties - may act as a signal through interactions with amino acid residues of the prokaryotic NER complex that are not possible with DNA. Such a mechanism might also be relevant if it were demonstrated that the eukaryotic NER machinery likewise incises an embedded ribonucleotide in DNA.

Dataset Information

The unstructured C-terminal extension of UvrD interacts with UvrB, but is dispensable for nucleotide excision repair.

Publications

The unstructured C-terminal extension of UvrD interacts with UvrB, but is dispensable for nucleotide excision repair.

Similar Datasets

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