Joyce L. Hamlin
Professor Emeritus
Ph.D., University of California, Los Angeles
Mechanisms of DNA Replication and Gene Amplification in Mammals

 

To facilitate analysis of a single replicon, we developed a methotrexate-resistant CHO cell line that has amplified a 240 kb sequence containing the dihydrofolate reductase locus approximately 1,000 times. We have shown by in vivo labelling studies that DNA replication in the amplicon initiates somewhere within the 55 kb region lying between the DHFR and 2BE2121 genes. This has been confirmed by several other origin-mapping approaches, including a two-dimensional gel electrophoretic technique that allows identification of the position along a template at which replication bubbles first form. Our data indicate that initiation sites are chosen from among a large number of potential sites scattered throughout the 55 kb intergenic region. We are investigating whether initiation in this origin is controlled by a genetic replicator, as it is in simpler organisms, or whether origin activity depends on localized transcriptional activity, nearby attachment to the nuclear scaffold, and/or higher order chromatin architecture. There are several genetic elements whose functions could contribute to the regulation of origin activity in this locus (e.g., a replicator, the promoters of the two flanking genes, the matrix attachment site). To identify these elements, we have devised a novel homologous recombination strategy to specificially knock out or mutagenize any fragment within the intergenic region or the two flanking genes. Our long-range goals are to define the initiation reaction in molecular terms by identifying both the cis- and trans-regulatory elements that participate in the reaction, and to reconstitute initiation in vitro.

We are also interested in the mechanism by which cells amplify DNA. DNA sequence amplification is an important phenomenon that only occurs in tumor cells, which usually lack critical damage-sensing pathways. Most human tumors have amplified one or more cellular oncogenes, which are thought to confer a selective growth advantage over surrounding normal cells. Thus, it is important to determine how gene amplification occurs. In recent fluorescence in situ hybridization studies, we have been able to show that the very first amplification events are mediated by chromosome breaks, followed by sister chromatid fusion, bridge formation, and further breaks. Thus, it is now clear why cells that lack the ability to sense DNA damage (i.e., breaks) are able to amplify oncogenes, while normal cells cannot. We are devising new strategies to examine the products of the earliest amplification events at the nucleotide sequence level to determine why and how cell breaks and fusions occur. Our goal is to identify steps in the amplification process that could be targets for chemotherapy.


Selected References

Mesner LD, Valsakumar V, Karnani N, Dutta A, Hamlin JL, Bekiranov S. (2011) "Bubble-chip analysis of human origin distributions demonstrates on a genomic scale significant clustering into zones and significant association with transcription." Genome Res. Jan [Epub ahead of print] [PubMed]

Hamlin JL, Mesner LD, Dijkwel PA. (2010) "A winding road to origin discovery." Chromosome Res. 18:45-61. [PubMed]

Mesner LD, Hamlin JL. (2009) "Isolation of restriction fragments containing origins of replication from complex genomes." Methods Mol Biol. 521:315-28. [PubMed]

Mesner LD, Dijkwel PA, Hamlin JL. (2009) "Purification of restriction fragments containing replication intermediates from complex genomes for 2-D gel analysis." Methods Mol Biol. 521:121-37. [PubMed]

Mesner LD, Crawford EL, Hamlin JL. (2006) "Isolating apparently pure libraries of replication origins from complex genomes." Mol Cell. Mar 21:719-26. [PubMed]

Mesner LD, Hamlin JL. (2005) "Specific signals at the 3' end of the DHFR gene define one boundary of the downstream origin of replication." Genes Dev. May 19:1053-66. [PubMed]

Alexandrow MG, Hamlin JL. (2005) "Chromatin decondensation in S-phase involves recruitment of Cdk2 by Cdc45 and histone H1 phosphorylation." J Cell Biol. Mar 168:875-86. Epub 2005 Mar 7. [PubMed]

Saha S, Shan Y, Mesner LD, Hamlin JL. (2004) "The promoter of the Chinese hamster ovary dihydrofolate reductase gene regulates the activity of the local origin and helps define its boundaries." Genes Dev. Feb 18:397-410. Epub 2004 Feb 20. [PubMed]

Mesner LD, Hamlin JL, Dijkwel PA. (2003) "The matrix attachment region in the Chinese hamster dihydrofolate reductase origin of replication may be required for local chromatid separation." Proc Natl Acad Sci U S A. Mar 100:3281-6. Epub 2003 Mar 10. [PubMed]