journal homepage: www.elsevier.com/locate/yexcr Available online at www.sciencedirect.com Research Article General and specific replication profiles are detected in normal human cells by genome-wide and single-locus molecular combing Elisa Palumbo 1 , Elena Tosoni 2 , Antonella Russo n Department of Biology, University of Padova, Via U. Bassi 58/b, 35131 Padova, Italy articleinformation Article Chronology: Received 28 March 2013 Received in revised form 24 September 2013 Accepted 1 October 2013 Keywords: DNA replication Mammalian cells Molecular combing abstract Mammalian genomes are replicated under a flexible program, with random use of origins and variable fork rates, and many details of the process must be still unraveled. Molecular combing provides a set of direct data regarding the replication profile of eukaryotic cells: fork rates; organization of the replication clusters; proportion of unidirectional forks; and fork dynamics. In this study the replication profiles of different primary and immortalized non-cancer human cells (lymphocytes, lymphoblastoid cells, fibroblasts) were evaluated at the whole-genome level or within reference genomic regions harboring coding genes. It emerged that these different cell types are characterized by specific replication profiles. In primary fibroblasts, a remarkable fraction of the mammalian genome was found to be replicated by unidirectional forks, and interestingly, the proportion of unidirectional forks further increased in the replicating genome along the population divisions. A second difference concerned in the proportion of paused replication forks, again more frequent in primary fibroblasts than in PBL/lymphoblastoid cells. We concluded that these patterns, whose relevance could escape when genomic methods are applied, represent normal replication features. In single-locus analyses, unidirectional and paused replication forks were highly represented in all genomic regions considered with respect to the average estimates referring to the whole-genome. In addition, fork rates were significantly lower than whole-genome estimates. Instead, when considering the specificities of each genomic region investigated (early to late replication, normal or fragile site) no further differentiating features of replication profiles were detected. These data, representing the integration of genome-wide and single-locus analyses, highlight a large heterogeneity of replication profiles among cell types and within the genome, which should be considered for the correct use of replication datasets. & 2013 Elsevier Inc. All rights reserved. 0014-4827/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2013.10.001 n Corresponding author. Fax: þ39 0 49 827 6280. E-mail addresses: elisa.palumbo@unipd.it (E. Palumbo), elena.tosoni@unipd.it (E. Tosoni), antonella.russo@unipd.it, russo@bio.unipd.it (A. Russo). 1 Present address: Familial Cancer Clinic and Oncoendocrinology, Veneto Institute of Oncology (IOV-IRCCS), Department of Medical and Surgical Sciences, University of Padova, Via Gattamelata 64, Padua, Italy. 2 Present address: Department of Molecular Medicine, University of Padova, Via A. Gabelli, 6, 35121 Padova, Italy. EXPERIMENTAL CELL RESEARCH ] ( ]]]] ) ]]] – ]]] Please cite this article as: E. Palumbo, et al., General and specific replication profiles are detected in normal human cells by genome- wide and single-locus molecular combing, Exp Cell Res (2013), http://dx.doi.org/10.1016/j.yexcr.2013.10.001