Conservation and Divergence in Nucleotide Excision Repair Lesion Recognition

Abstract

Nucleotide excision repair is an important and highly conserved DNA repair mechanism with an exceptionally large range of chemically and structurally unrelated targets. Lesion verification is believed to be achieved by the helicases UvrB and XPD in the prokaryotic and eukaryotic processes, respectively. Using single molecule atomic force microscopy analyses, we demonstrate that UvrB and XPD are able to load onto DNA and pursue lesion verification in the absence of the initial lesion detection proteins. Interestingly, our studies show different lesion recognition strategies for the two functionally homologous helicases, as apparent from their distinct DNA strand preferences, which can be rationalized from the different structural features and interactions with other nucleotide excision repair protein factors of the two enzymes. Nucleotide excision repair (NER)4 is an important DNA repair mechanism with a large range of chemically and structurally unrelated targets. Examples range from bulky DNA adducts to interstrand DNA cross-links caused by antitumor drugs (1-3). In humans, NER is the only repair system for the removal of UV irradiation-induced photoproducts such as the intrastrand cross-linking cyclobutane-pyrimidine dimers (CPDs), and dysfunctional NER is responsible for severe diseases, including xeroderma pigmentosum (XP) (1-3).The mechanism of NER is highly conserved between organisms. In bacteria, NER involves the proteins UvrA, UvrB, and UvrC. In the current model of prokaryotic NER, a hetero-tetrameric complex of UvrA and UvrB (UvrA 2 B 2 ) scans the DNA for lesions. When a lesion is encountered, initial lesion sensing by a dimer of UvrA is based on detection of DNA distortion. Conformational changes in the UvrA 2 B 2 complex result in an unwinding and opening of dsDNA around the lesion, providing an unpaired (bubble) region likely required by UvrB to thread onto one of the ssDNA strands. The helicase UvrB is believed to verify the presence of a lesion by insertion of a ␤-hairpin via interactions with residues at the base of the hairpin (2). Upon lesion verification by UvrB, UvrA dissociates from the complex, and ATP re-binding by UvrB results in the formation of the lesion-specific UvrB-DNA complex, which recruits the NER endonuclease UvrC (UvrBC complex). UvrC carries out two incisions on either side of the lesion. The 12-13-nucleotide (nt)-long ssDNA stretch (2) containing the lesion can then be removed together with the endonuclease by the helicase UvrD, and the resulting gap is filled and sealed by DNA polymerase I and ligase.Eukaryotic NER encompasses a total of ϳ30 proteins, including the xeroderma pigmentosum group proteins (XPA-XPG). Repair can either be initiated by a stalled RNA polymerase in transcription-coupled NER or via global genome NER. In global genome NER, upon initial detection of short destabilized DNA structures by the CEN2-XPC-HR23B complex, the ATPase/ helicase XPB, which is part of the 10-subunit transcription factor IIH (TFIIH) complex, then directly interacts with XPC (4) and...

Recruitment of UvrBC complexes to UV-induced damage in the absence of UvrA increases cell survival

“…Numerous complexes have been proposed to form during NER in prokaryotes; however, their dynamics and specific roles remain uncertain. Single-molecule and ensemble approaches have been used previously to reveal the existence of UvrAB ( 4 , 6 , 9 , 11 ) and UvrBC ( 19 , 20 , 22–24 ) complexes, which likely represent the most populated forms in vivo . The UvrAB complex is well established in damage search; however a role for the UvrBC complex in a process other than DNA incision is not clear.…”

“…Our observations suggested, in agreement with bulk studies ( 9 , 13 ), that UvrA and UvrB formed a UvrA 2 B 2 complex. In addition, UvrB and UvrC were also observed to form a complex independently of UvrA ( 19 , 20 ). This intriguing complex has been previously identified during bulk studies ( 21 , 22 ), but has shown no apparent repair efficiency on dsDNA in vitro ( 20 , 23 , 24 ).…”

Mechanistic and biological considerations of oxidatively damaged DNA for helicase-dependent pathways of nucleic acid metabolism

“…Moreover, it has been postulated that certain helicases (e.g., XPB, XPD) play a role in the recognition or verification of bulky DNA damage [26–31]. It is less certain but plausible that other helicases (e.g., RecQ members) may sense DNA damage as a component of the replication stress response [32].…”

Mechanistic insights into transcription coupled DNA repair

“…To maintain the balance required for survival and diversity, evolution has equipped all life forms with mechanisms to either eliminate or tolerate DNA damage. Basic strategies for DNA repair include removing individual damaged nucleotides via the base excision repair (BER) mechanism [4] or by excising longer sections containing helix-distorting bulky lesions through the nucleotide excision repair (NER) mechanism[5]. …”