In eukaryotic cells, DNA in each chromosome is synthesized by multiple tandemly organized replication units (replicons), which are activated at precisely defined times of the S phase. In each replicon, initiation of DNA replication occurs at a specific site (origin of DNA replication) from which two oppositely moving semiconservative forks originate. The concept that DNA synthesis in metazoan chromosomes initiates at preferred sites is supported by several different studies. However, at present, there is no reliable functional assay for origin activity. Because of this experimental limitation, a variety of physico-chemical or biochemical approaches have been developed for mapping the localization of DNA replication origins directly in the mammalian genome. Most of these studies, however, entail the utilization of potentially artifactual techniques (such as utilization of protein inhibitors, cell synchronization, or cell permeabilization) or produce results which are often difficult to interpret (such as 2D gel analysis). The work presented in this thesis has been an effort to understand initiation of DNA replication in mammalian cells. For this purpose, I contributed to the development of a novel procedure for mapping origins of DNA replication, based on the quantification of nascent DNA strands by competitive PCR. The research has been initially focused on Chinese hamster cells with the prospect to precisely identify the origin region in the DHFR locus. With this regard, the hamster DHFR origin region is particularly challenging since virtuallly every available mapping approach has been utilized to investigate it. However, despite these intense investigations, contradictions on the nature of DNA replication origins in this region are still unexplained. By using the origin mapping protocol we developed, a high-resolution mapping study on the single copy DHFR gene locus in hamster CHO K1 cells has been performed. The results obtained reinforce the notion that DNA replication starts at a precise site, located -17 kb downstream of the DHFR gene. In the nascent DNA samples, the fragment containing the start site is enriched over 10-fold with respect to those located at the two boundaries of the analyzed region, or within the 31 end of the DHFR gene itself. The origin region has been trimmed down to a -800 bp segment. Further, our experience in nascent DNA analysis, increased by an intense search for technical improvements and comprehension of the mapping technique, has encouraged its extensive application for the identification of other origins in mammalian chromosomes. However, since the systematic analysis of large regions of mammalian chromosomes by PCR quantification of nascent DNA is highly demanding and time consuming, we have attempted to develop a new approach for the identification of DNA replication origins. For this purpose, I constructed a library of replication origin-enriched sequences from human cells to be used as a probe for hybridization to immobilized cloned fragments from a region of interest. The principle of the method is that, being the library enriched in origin sequences, it will preferentially hybridize to immobilized fragments containing an origin of replication. This approach, when applied to the human lamin 82 region where an origin of replication had already been mapped by quantification of nascent strand abundance by competitive PCR, has allowed to identify in a very simple and rapid way the origin. Library construction and characterization are presented in the second part of the thesis, together with its possible usage to identify new origins of DNA replication in human cells. Finally, with the purpose to study factors binding to and activating origins, recombinant proteins and antibodies useful in this respect have been prepared. Since work on this topic is still ongoing in the laboratory, preparation of these reagents is presented as an appendix to the thesis.
|Titolo:||Analysis of Nascent DNA as a Tool for the Identification of Mammalian Origins of DNA Replication|
|Relatore/i esterni:||Falaschi, Arturo|
|Data di pubblicazione:||15-dic-1997|
|Appare nelle tipologie:||8.1 PhD thesis|