DNA mismatch binding activities in Chlorella pyrenoidosa extracts and affinity isolation of G-T mismatch binding proteins.
DNA mismatch recognition proteins contained in the extracts of unicellular alga Chlorella pyrenoidosa were isolated by affinity adsorption and 2-D gel electrophoresis. Incubation of the algal extracts with a 38-mer duplex oligonucleotide carrying a single DNA simple mispair generated a few gel retardation complexes. G-T mispair was recognized significantly better than C-T, G-G, G-A, and C-C mispairs by the algal extracts and these extracts bound very weakly to G-A and C-C mispairs, displaying a universal trend of mismatch binding efficiency. The levels of mismatch recognition complexes were slightly increased in the presence of 1 mM ATP. Two 13-kDa G-T binding polypeptides possessing pIs of 5.3 and 5.5 were isolated after resolving affinity-captured proteins by 2-D gel electrophoresis and the two factors were found to bind 5.5- and 2.8-fold stronger to heteroduplex than to homoduplex DNA, respectively. No proteins significantly homologous to the two algal G-T binding proteins were found by peptide mass fingerprinting (PMF). The sequence of a peptide generated from trypsin-cleavage of one G-T binding factor (pI 5.5) could be aligned with the amino acid sequences that form the C-terminal active sites of human and mouse mismatch-specific uracil/thymine-DNA glycosylases, suggesting the possibility of this factor as an algae- or a Chlorella-specific DNA mismatch glycosylase.
Hsu
Todd
T
Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan, ROC. toddhsu@mail.ntou.edu.tw
Chang
Kai-Ning
KN
Lai
Yi-Show
YS
Jung
Ting-Yi
TY
Lee
Gen-I
GI
D020418:Algal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D020137:Base Pair Mismatch; D002708:Chlorella / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D024202:Electrophoretic Mobility Shift Assay; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D017386:Sequence Homology, Amino Acid
D020418:Algal Proteins; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D004247:DNA