The DHFR origin consists of a zone of >22 inefficient initiation sites scattered throughout the 55 kb spacer between the convergently-transcribed DHFR and 2BE2121 genes. However, some sites (ori-? and ori-?) are clearly preferred. We have shown that in loco deletion of the most active initiation sites from the DHFR origin, or even the central 40-kb core that encompasses >90% of active sites, does not suppress initiation in the remainder of the spacer. In fact, initiation frequency actually increases. Thus, master replicators do not reside in the origin itself. However, an active DHFR promoter is required for maximum origin activity and, in its absence, replication initiates in the now-inactive gene. Furthermore, the 3'processing signals of the gene prevent inactivation of the origin by read-through transcription. We propose that mammalian genomes are peppered at intervals of <1 kb with degenerate replicators whose activities are modulated by local transcription and chromatin architecture.
Specific aims are: 1) to determine whether the human lamin B2 and ?-globin origins, which are thought to represent true replicators, are intrinsically more active than the most active sites in the DHFR initiation zone;ori-? will be flanked with mismatched LoxP sites and exchanged with the two human origins, or with fragments that do not initiate efficiently in loco;the activities of each will then be quantified;lamin B2 and ?-globin also will be compared in human cells to fragments residing in initiation zones;2) to determine whether ori-? and ori-? are more active than other sites in the spacer because of their locations vis-?-vis the local transcription units;ori-? will be positioned differently in the spacer by the ROKO approach, and effects on origin efficiency assessed;3) to determine whether DHFR and the weaker 2BE2121 promoters play essential but redundant roles in origin activation;both promoters will be deleted, and effects on origin activity will be assessed;in addition, a strong transcription stop signal will be placed just downstream from the DHFR promoter to determine whether transcription fork movement (as opposed to the promoter per se) is required for origin stimulation;4) to determine why the efficiency of initiation increases when the intergenic spacer is truncated, and to prepare a """"""""super"""""""" origin;the length of the spacer will be reduced to determine optimal size and a strong transcription terminator will be placed downstream from the DHFR and 2BE2121 genes;5) to define the epigenetic changes that occur when ori-? is inactivated by deletion of 5'and 3'regulatory elements or activated by one of the above strategies;ChIP technology will be utilized to determine the distributions of initiation proteins and modified histones as a function of the cell cycle in variants elaborated in Aims 1-4;these studies will indicate factors that limit origin efficiency, and will test the predictions that transcription through the spacer removes pre-RCs, while promoter deletions result in a failure to load them.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026108-32
Application #
8099533
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Janes, Daniel E
Project Start
1978-09-01
Project End
2014-06-30
Budget Start
2011-07-01
Budget End
2014-06-30
Support Year
32
Fiscal Year
2011
Total Cost
$440,461
Indirect Cost
Name
University of Virginia
Department
Biochemistry
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Mesner, Larry D; Dijkwel, Pieter A; Hamlin, Joyce L (2015) Purification of restriction fragments containing replication intermediates from complex genomes for 2-D gel analysis. Methods Mol Biol 1300:261-77
Mesner, Larry D; Valsakumar, Veena; Cieslik, Marcin et al. (2013) Bubble-seq analysis of the human genome reveals distinct chromatin-mediated mechanisms for regulating early- and late-firing origins. Genome Res 23:1774-88
Wong, Philip G; Winter, Sherry L; Zaika, Elena et al. (2011) Cdc45 limits replicon usage from a low density of preRCs in mammalian cells. PLoS One 6:e17533
Mesner, Larry D; Valsakumar, Veena; Karnani, Neerja et al. (2011) Bubble-chip analysis of human origin distributions demonstrates on a genomic scale significant clustering into zones and significant association with transcription. Genome Res 21:377-89
Hamlin, Joyce L; Mesner, Larry D; Dijkwel, Pieter A (2010) A winding road to origin discovery. Chromosome Res 18:45-61
Mesner, Larry D; Dijkwel, Pieter A; Hamlin, Joyce L (2009) Purification of restriction fragments containing replication intermediates from complex genomes for 2-D gel analysis. Methods Mol Biol 521:121-37
Mesner, Larry D; Hamlin, Joyce L (2009) Isolation of restriction fragments containing origins of replication from complex genomes. Methods Mol Biol 521:315-28
Hamlin, J L; Mesner, L D; Lar, O et al. (2008) A revisionist replicon model for higher eukaryotic genomes. J Cell Biochem 105:321-9
Czajkowsky, Daniel M; Liu, Jie; Hamlin, Joyce L et al. (2008) DNA combing reveals intrinsic temporal disorder in the replication of yeast chromosome VI. J Mol Biol 375:12-9
Mesner, Larry D; Hamlin, Joyce L (2005) Specific signals at the 3' end of the DHFR gene define one boundary of the downstream origin of replication. Genes Dev 19:1053-66

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