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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...

In eukaryotes, DNA methylation is an important epigenetic modification that is generally involved in gene regulation. Methyltransferases (MTases) of the DNMT2 family have been shown to have a dual substrate specificity acting on DNA as well as on three specific tRNAs (tRNAAsp, tRNAVal, tRNAGly). Entamoeba histolytica is a major human pathogen, and expresses a single DNA MTase (EhMeth) that belongs to the DNMT2 family and shows high homology to the human enzyme as well as to the bacterial DNA MTase M.HhaI. The molecular basis for the recognition of the substrate tRNAs and discrimination of non-cognate tRNAs is unknown. Here we present the crystal structure of the cytosine-5-methyltransferase EhMeth at a resolution of 2.15 Å, in complex with its reaction product S-adenosyl-L-homocysteine, revealing all parts of a DNMT2 MTase, including the active site loop. Mobility shift assays show that in vitro the full length tRNA is required for stable complex formation with EhMeth.

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The helicases XPB and XPD are part of the TFIIH complex, which mediates transcription initiation as well as eukaryotic nucleotide excision repair (NER). Although there is no TFIIH complex present in archaea, most species contain both XPB and XPD and serve as a model for their eukaryotic homologs. Recently, a novel binding partner for XPB, Bax1 (binds archeal XPB), was identified in archaea. To gain insights into its role in NER, Bax1 from Thermoplasma acidophilum was characterized. We identified Bax1 as a novel Mg 2؉ -dependent structurespecific endonuclease recognizing DNA containing a 3 overhang. Incision assays conducted with DNA substrates providing different lengths of the 3 overhang indicate that Bax1 specifically incises DNA in the single-stranded region of the 3 overhang 4 -6 nucleotides to the single-stranded DNA/doublestranded DNA junction and thus is a structure-specific and not a sequence-specific endonuclease. In contrast, no incision was detected in the presence of a 5 overhang, double-stranded DNA, or DNA containing few unpaired nucleotides forming a bubble. Several Bax1 variants were generated based on multiple sequence alignments and examined with respect to their ability to perform the incision reaction. Residues Glu-124, Asp-132, Tyr-152, and Glu-155 show a dramatic reduction in incision activity, indicating a pivotal role in catalysis. Interestingly, Bax1 does not exhibit any incision activity in the presence of XPB, thus suggesting a role in NER in which the endonuclease activity is tightly regulated until the damage has been recognized and verified prior to the incision event. Nucleotide excision repair (NER)2 is a DNA repair mechanism that is responsible for the removal of a vast diversity of bulky DNA damages (1). Failures in the NER pathway can ultimately lead to cancer, as observed in the skin cancer-prone disease xeroderma pigmentosum (XP). In addition, because several chemotherapeutic agents are recognized and repaired by NER, the underlying mechanism of damage recognition and repair is an important issue in cancer treatment (2).The helicases XPB and XPD are part of the TFIIH complex, which mediates eukaryotic NER as well as transcription initiation. Mutations in either protein lead to severe diseases such as XP, Cockayne syndrome, or trichothiodystrophy. Although there is no TFIIH complex present in archaea, most species contain both XPB and XPD and serve as a model for their eukaryotic homologs (3). It is important to note that the structure from Thermoplasma acidophilum XPD, which was recently solved, could explain the effect of several point mutations leading to the phenotype of patients suffering from XP, trichothiodystrophy, or XP/Cockayne syndrome (4 -6).In contrast to the active helicase XPD, XPB exerts only limited helicase activity (7,8). However, its ATPase activity was shown to be crucial for TFIIH to unwind DNA (8). Thus, it is assumed that XPD, the major helicase in TFIIH, is responsible for further strand opening during the process of NER. Taken together, these data sug...