Mapping of replication initiation sites in human ribosomal DNA by nascent-strand abundance analysis.
New techniques for mapping mammalian DNA replication origins are needed. We have modified the existing nascent-strand size analysis technique (L. Vassilev and E.M. Johnson, Nucleic Acids Res. 17:7693-7705, 1989) to provide an independent means of studying replication initiation sites. We call the new method nascent-strand abundance analysis. We confirmed the validity of this method with replicating simian virus 40 DNA as a model. We then applied nascent-strand abundance and nascent-strand size analyses to mapping of initiation sites in human (HeLa) ribosomal DNA (rDNA), a region previously examined exclusively by two-dimensional gel electrophoresis methods (R.D. Little, T.H.K. Platt, and C.L. Schildkraut, Mol. Cell. Biol. 13:6600-6613, 1993). Our results partly confirm those obtained by two-dimensional gel electrophoresis techniques. Both studies suggest that replication initiates at relatively high frequency a few kilobase pairs upstream of the transcribed region and that many additional low-frequency initiation sites are distributed through most of the remainder of the ribosomal DNA repeat unit.
Yoon
Y
Y
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Sanchez
J A
JA
Brun
C
C
Huberman
J A
JA
D000818:Animals; D002460:Cell Line; D002874:Chromosome Mapping / Q000379:methods*; D015342:DNA Probes; D004261:DNA Replication / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D018741:Replication Origin / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics
D015342:DNA Probes; D004275:DNA, Ribosomal