Identification and sequence characterization of a 1.3 Kb EcoRI repeat fragment that harbors a DNA repair site of rat pachytene spermatocytes.
Introduction of well-programmed nicks and gaps and the associated DNA repair activity in the genome at the pachytene interval is a characteristic feature of the meiotic prophase in organisms as varied as lilium and mouse. In the present study we have shown that the DNA synthetic activity in rat pachytene spermatocytes is insensitive to aphidicolin, a specific inhibitor of DNA polymerase alpha, delta and epsilon, suggesting DNA beta-polymerase-mediated repair synthesis in these cells. We have developed a novel approach for the isolation of the DNA repair sites by combining two independent techniques. Following incorporation of BrdUrd into pachytene spermatocytes in the presence of aphidicolin, the repair sites were released as ssDNA fragments by treatment of nuclei with 30 mM NaOH. Subsequently, the BrdUrd containing ssDNA fragments were specifically isolated using polyclonal anti-BrdUrd antibodies. The DNA fragments released were of two size classes, namely 4-7S (major) and 9-12S (minor) and constituted approximately 1.75% of the pachytene genomic DNA. These DNA repair fragments were distinct from Okazaki fragments and other replicative intermediates isolated from rat bone marrow cells as evidenced by (a) their different size distribution and (b) little cross-hybridization. Southern hybridization of restriction enzyme digests of rat genomic DNA with probes made against BrdUrd-ssDNA fragments revealed that although the repair sites were distributed throughout the genome, strong hybridization signals were observed in EcoRI. (1.3 kb and 2.4 kb), BamH1 (9 kb) and HindIII (5 kb) repetetive DNA fragments. The EcoRI 1.3 kb family were cloned into M13 mp19, and a repair positive (1.3 A) and a repair negative (1.3 B) were identified and sequenced. The repair positive clone contained (a) (CA)22 repeat, (b) a (CAGA)6 repeat and (c) 4 sequences sharing high homology with various hypervariable minisatellite (HVMS) sequences. One of the HVMS sequence contained a GGCAGG motif known to be responsible for germline instability. The repair negative clone had (a) (CA)6 repeat and (b) a HVMS like sequence without GGCAGG. The significance of these motifs and their relevance to the events of DNA metabolism at pachytene interval have been discussed.
Ramachandra
L
L
Department of Biochemistry, Indian institute of Science, Bangalore.
Rao
M R
MR
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D001483:Base Sequence; D001853:Bone Marrow / Q000187:drug effects; D001854:Bone Marrow Cells; D004256:DNA Polymerase I / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects; D015246:Deoxyribonuclease EcoRI; D015723:Gene Library; D006801:Humans; D008297:Male; D008540:Meiosis; D018598:Minisatellite Repeats; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012150:Polymorphism, Restriction Fragment Length; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013087:Spermatids / Q000378:metabolism; D013090:Spermatocytes / Q000378:metabolism*
D016590:Aphidicolin; D004256:DNA Polymerase I; D015246:Deoxyribonuclease EcoRI