Start Sites of Bidirectional DNA Synthesis at the Human Lamin B2 Origin

Abstract

The initiation sites of bidirectional synthesis at the DNA replication origin located at the 3' end of the human lamin B2 gene were investigated. RNA-primed nascent DNA molecules were subjected to second-strand synthesis with appropriate primers, amplified by ligation-mediated polymerase chain reaction, and size fractionated. Evidence for precise start sites was obtained. Exploration of close to 1 kilobase, coupled to inhibition of Okazaki fragment synthesis, demonstrates that the leading strands initiate at precise nucleotides on either helix, overlapping by three base pairs, within the area bound to a protein complex possibly analogous to the prereplicative complex of yeast.

One-way PCR-based mapping of a replication initiation point (RIP)

“…In yeast cells, one strategy to circumvent this problem is to compare RIPs in wild-type and DNA ligase I-deficient mutants 3 . Since this method is difficult to apply in mammalian cells, we and others used emetine, which inhibits new production of lagging strands without affecting leading strand synthesis 1,11 . Although concern was raised previously regarding drug treatments and the generation of potential artifactual results 2 , we did not observe any notable problemwhenwe determined DBF4 RIPs with cells maintained in the presence and absence of emetine 10 .…”

“…Choice of DNA polymerases-Although Vent (exo − ) DNA polymerase has been the polymerase of choice for RIP mapping [1][2][3] , this enzyme was not ideal under our experimental conditions. The possible reason for this is that the activity of this polymerase may not be compatible with betaine.…”

“…However, RIP mapping in mammalian cells turned out to be very challenging, mainly due to technical difficulties. The lamin B2 locus is thus far the only mammalian ori whose RIPs have been mapped 1 . The authors determined RIPs at this locus using the technically demanding ligation-mediated (LM) PCR 1 .…”

“…The lamin B2 locus is thus far the only mammalian ori whose RIPs have been mapped 1 . The authors determined RIPs at this locus using the technically demanding ligation-mediated (LM) PCR 1 . Although this paper was reported in 2000, no one has reported another mammalian RIP using LM-PCR or any other methods, underscoring the need to develop more effective and user friendly RIP-mapping techniques.…”

“…However, the original RIP-mapping protocol was not considered sensitive enough to map RIPs in mammalian cells. Therefore, LM-PCR was used to map RIP at the human lamin B2 origin 1 .…”