High-Resolution Analysis of DNA Replication Domain Organization across an R/G-Band Boundary

Abstract

Establishing how mammalian chromosome replication is regulated and how groups of replication origins are organized into replication bands will significantly increase our understanding of chromosome organization. Replication time bands in mammalian chromosomes show overall congruency with structural R-and Gbanding patterns as revealed by different chromosome banding techniques. Thus, chromosome bands reflect variations in the longitudinal structure and function of the chromosome, but little is known about the structural basis of the metaphase chromosome banding pattern. At the microscopic level, both structural R and G bands and replication bands occupy discrete domains along chromosomes, suggesting separation by distinct boundaries. The purpose of this study was to determine replication timing differences encompassing a boundary between differentially replicating chromosomal bands. Using competitive PCR on replicated DNA from flow-sorted cell cycle fractions, we have analyzed the replication timing of markers spanning roughly 5 Mb of human chromosome 13q14.3/q21.1. This is only the second report of high-resolution analysis of replication timing differences across an R/G-band boundary. In contrast to previous work, however, we find that band boundaries are defined by a gradient in replication timing rather than by a sharp boundary separating R and G bands into functionally distinct chromatin compartments. These findings indicate that topographical band boundaries are not defined by specific sequences or structures.Human metaphase chromosomes can be cytogenetically identified by a distinctive banding pattern that is achieved by different staining techniques. Chromosome banding using DNA-specific stains such as Giemsa and quinacrine was first described almost 30 years ago (42), and the most common banding types are now referred to as Giemsa (G) bands and reverse (R) bands. Despite significant advances in staining techniques, there has been little progress in understanding the underlying chromosomal structures responsible for the banding phenomenon. R bands are characterized by early replication, high gene and CpG island density, and localization of a large number of both housekeeping and tissue-specific genes, and they are enriched for short interspersed repetitive elements. G bands are late replicating, contain a low number of tissue-specific genes, and are enriched for long interspersed repetitive elements (5,6,11,19,20,31,32). These general properties of R and G bands define them as distinct structural and functional units, suggesting the presence of discrete topographical boundaries. However, at higher levels of band resolution, it seems to be increasingly difficult to define narrow chromosome band boundaries, and the potential to correlate specific bands with specific functional sequences seems unlikely (19). Thus, to understand how the genome is organized in alternating chromosome bands and how boundaries between them are established, it is important to analyze functional differences between bands at the mole...

Isochores and replication time zones: a perfect match

“…This finding is further supported by the results for the second late replicating domain within the MN1 / PITPNB gene region: Pool 6 that covers about 100 kb of DNA from the late replicating isochore D is probably replicated by one single replication fork; hence its replication takes about 30 min. In contrast to this, interphase FISH analysis revealed a difference of three hours in the replication timing of isochores C and D. Therefore, our results contradict a gradual transition between early and late replicated domains as it has been reported for switches between replication time zones on human chromosome 13q14.3/ q21.1 (Strehl et al, 1997) and at the IgH locus of the mouse (Ermakova et al, 1999).…”

Replication Delay along FRA7H, a Common Fragile Site on Human Chromosome 7, Leads to Chromosomal Instability

“…Thus, a similar replication time pattern is generally expected over extensive (megabases) genomic regions until a boundary between replication zones is reached (38). Upon aphidicolin treatment, this regulation is not expected to change.…”

Identification of a cis-Element That Determines Autonomous DNA Replication in Eukaryotic Cells

“…Although homologies could be observed for stretches of sequence within initiation regions of defined origins of replication, the length of homology and degree of similarity were not complete. We have been successful in using the consensus sequence to predict probable regions that may contain autonomous replication activity and origins at the ␥-aminobutyric acid receptor subunit ␤3 and ␣5 gene cluster (45,46) and at the dnmt1 (human DNA methyltransferase) locus, wherein binding to Ku86 was also verified (47). In a FASTA search of the GenBank data base, very significant homology was observed with certain CpG island clones (30), up to 89% over 36 bp and with the allowance of a single base gap, 100% over 36 bp.…”