title: Modulation of the processive abasic site lyase activity of a pyrimidine dimer glycosylase.
abstract: The repair of cis-syn cyclobutane pyrimidine dimers (CPDs) can be initiated via the base excision repair (BER) pathway, utilizing pyrimidine dimer-specific DNA glycosylase/lyase enzymes (pdgs). However, prior to incision at lesion sites, these enzymes bind to non-damaged DNAs through charge-charge interactions. Following initial binding to DNA containing multiple lesions, the enzyme incises at most of these sites prior to dissociation. If a subset of these lesions are in close proximity, clustered breaks may be produced that could lead to decreased cell viability or increased mutagenesis. Based on the co-crystal structures of bacteriophage T4-pdg and homology modeling of a related enzyme from Paramecium bursaria Chlorella virus-1, the structure-function basis for the processive incision activity for both enzymes was investigated using site-directed mutagenesis. An assay was developed that quantitatively measured the rates of incision by these enzymes at clustered apurinic/apyrimidinic (AP) sites. Mathematical modeling of random (distributive) versus processive incisions predicted major differences in the rate and extent of the accumulation of singly nicked DNAs between these two mechanisms. Comparisons of these models with biochemical nicking data revealed significant changes in the damage search mechanisms between wild-type pdgs and most of the mutant enzymes. Several conserved arginine residues were shown to be critical for the processivity of the incision activity, without interfering with catalysis at AP sites. Comparable results were measured for incision at clustered CPD sites in plasmid DNAs. These data reveal that pdgs can be rationally engineered to retain full catalytic activity, while dramatically altering mechanisms of target site location.
authors: Center for Research on Occupational and Environmental Toxicology, Oregon Health & Science University, Portland, OR 97239, USA.
authors: Olga P
authors: OP
authors: Ryabinina
authors: Irina G
authors: IG
authors: Minko
authors: Michael R
authors: MR
authors: Lasarev
authors: Amanda K
authors: AK
authors: McCullough
authors: R Stephen
authors: RS
authors: Lloyd
mesh_terms: D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology*; D002384:Catalysis; D053904:DNA Breaks, Single-Stranded; D004249:DNA Damage / Q000235:genetics; D045647:DNA Glycosylases / Q000737:chemistry* / Q000235:genetics; D011088:DNA Ligases / Q000737:chemistry / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D008190:Lyases / Q000737:chemistry* / Q000235:genetics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011740:Pyrimidine Dimers / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D011740:Pyrimidine Dimers; D014764:Viral Proteins; D045647:DNA Glycosylases; C036401:deoxyribopyrimidine endonucleosidase; D008190:Lyases; C110456:Chlorella virus DNA ligase; D011088:DNA Ligases