Bax1 is a novel endonuclease: implications for archaeal nucleotide excision repair.
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 structure-specific 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/double-stranded 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.
Roth
Heide M
HM
Rudolf-Virchow-Center for Experimental Biomedicine, 97080 Würzburg, Germany.
Tessmer
Ingrid
I
Van Houten
Bennett
B
Kisker
Caroline
C
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D007477:Ions; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013822:Thermoplasma / Q000378:metabolism
D019843:Archaeal Proteins; D004277:DNA, Single-Stranded; D007477:Ions; D009711:Nucleotides; D004247:DNA; D004706:Endodeoxyribonucleases; D008274:Magnesium