Biomedical subjects

Ronald Lebofsky

Publications and source records attributed to Ronald Lebofsky.

4 recordsLinked to original sources

PubMed2006

DNA replication origin interference increases the spacing between initiation events in human cells.

Mammalian DNA replication origins localize to sites that range from base pairs to tens of kilobases. A regular distribution of initiations in individual cell cycles suggests that only a limited number of these numerous potential start sites are converted into activated origins. Origin interference can silence redundant origins; however, it is currently unknown whether interference participates in spacing functional human initiation events. By using a novel hybridization strategy, genomic Morse code, on single combed DNA molecules from primary keratinocytes, we report the initiation sites present on 1.5 Mb of human chromosome 14q11.2. We confirm that initiation zones are widespread in human cells, map to intergenic regions, and contain sequence motifs found at other mammalian initiation zones. Origins used per cell cycle are less abundant than the potential sites of initiation, and their limited use increases the spacing between initiation events. Between-zone interference decreases in proportion to the distance from the active origin, whereas within-zone interference is 100% efficient. These results identify a hierarchical organization of origin activity in human cells. Functional origins govern the probability that nearby origins will fire in the context of multiple potential start sites of DNA replication, and this is mediated by origin interference.

Base Pairing↗

PubMed2005

Human ribosomal RNA gene arrays display a broad range of palindromic structures.

The standard model of eukaryotic ribosomal RNA (rRNA) genes involves tandem arrays with hundreds of units in clusters, the nucleolus organizer regions (NORs). A first genomic overview for human cells is reported here for these regions, which have never been sequenced in their totality, by using molecular combing. The rRNA-coding regions are examined by fluorescence on single molecules of DNA with two specific probes that cover their entire length. The standard organization assumed for rDNA units is a transcribed region followed by a nontranscribed spacer. While we confirmed this arrangement in many cases, unorthodox patterns were also observed in normal individuals, with one-third of the rDNA units rearranged to form apparently palindromic structures (noncanonical units) independent of the age of the donors. In cells from individuals with a deficiency in the WRN RecQ helicase (Werner syndrome), the proportion of palindromes increased to one-half. These findings, supported by Southern blot analyses, show that rRNA genes are a mosaic of canonical and (presumably nonfunctional) palindromic units that may be altered by factors associated with genomic instability and pathology.

Blotting, Southern↗

PubMed2005

DNA replication origin plasticity and perturbed fork progression in human inverted repeats.

The stability of metazoan genomes during their duplication depends on the spatiotemporal activation of origins and the progression of forks. Human rRNA genes represent a unique challenge to DNA replication since a large proportion of them exist as noncanonical palindromes in addition to canonical tandem repeats. Whether origin usage and/or fork elongation can cope with the variable structure of these genes is unknown. By analyzing single combed DNA molecules from HeLa cells, we studied the rRNA gene replication program according to the organization of canonical versus noncanonical rRNA genes. Origin positioning, spacing, and timing were not affected by the underlying rRNA gene physical structure. Conversely, fork arrest, both temporary and permanent, occurred more frequently when rRNA gene palindromes were encountered. These findings reveal that while initiation mechanisms are flexible enough to adapt to an rRNA gene structure of any arrangement, palindromes represent obstacles to fork progression, which is a likely source of genomic instability.

DNA Replication↗

PubMed2003

Single DNA molecule analysis: applications of molecular combing.

Dynamic changes to the genomic structure and to the DNA replication programme are important determinants of normal and abnormal cell development. To understand these changes and how they vary from cell to cell, single DNA molecules from both normal and abnormal cell populations must be examined and compared. Physical characterisation of single genomes at the kilobase level of resolution over large genomic regions is possible with molecular combing technology. An array of combed single DNA molecules is prepared by stretching molecules attached by their extremities to a silanised glass surface with a receding air-water meniscus. By performing fluorescent hybridisation on combed DNA, genomic probe position can be directly visualised, providing a means to construct physical maps and detect micro-rearrangements. Single-molecule DNA replication can also be monitored through fluorescent detection of incorporated nucleotide analogues on combed DNA molecules. These and other single-molecule applications of molecular combing are discussed in this paper and future developments of the technology are considered.

DNA↗