To maintain genomic stability, reinitiation of eukaryotic DNA replication within a single cell cycle is blocked by multiple mechanisms that inactivate or remove replication proteins after G1 phase. Consistent with the prevailing notion that these mechanisms are redundant, we previously showed that simultaneous deregulation of three replication proteins, ORC, Cdc6, and Mcm2-7, was necessary to cause detectable bulk re-replication in G2/M phase in Saccharomyces cerevisiae. In this study, we used microarray comparative genomic hybridization (CGH) to provide a more comprehensive and detailed analysis of re-replication. This genome-wide analysis suggests that reinitiation in G2/M phase primarily occurs at a subset of both active and latent origins, but is independent of chromosomal determinants that specify the use and timing of these origins in S phase. We demonstrate that re-replication can be induced within S phase, but differs in amount and location from re-replication in G2/M phase, illustrating the dynamic nature of DNA replication controls. Finally, we show that very limited re-replication can be detected by microarray CGH when only two replication proteins are deregulated, suggesting that the mechanisms blocking re-replication are not redundant. Therefore we propose that eukaryotic re-replication at levels below current detection limits may be more prevalent and a greater source of genomic instability than previously appreciated.

The human DEK gene is frequently overexpressed and sometimes amplified in human cancer. Consistent with oncogenic functions, Dek knockout mice are partially resistant to chemically induced papilloma formation. Additionally, DEK knockdown in vitro sensitizes cancer cells to DNA damaging agents and induces cell death via p53-dependent and -independent mechanisms. Here we report that DEK is important for DNA double-strand break repair. DEK depletion in human cancer cell lines and xenografts was sufficient to induce a DNA damage response as assessed by detection of γH2AX and FANCD2. Phosphorylation of H2AX was accompanied by contrasting activation and suppression, respectively, of the ATM and DNA-PK pathways. Similar DNA damage responses were observed in primary Dek knockout mouse embryonic fibroblasts (MEFs), along with increased levels of DNA damage and exaggerated induction of senescence in response to genotoxic stress. Importantly, Dek knockout MEFs exhibited distinct defects in non-homologous end joining (NHEJ) when compared to their wild-type counterparts. Taken together, the data demonstrate new molecular links between DEK and DNA damage response signaling pathways, and suggest that DEK contributes to DNA repair.

Overexpression of the DEK gene is associated with multiple human cancers, but its specific roles as a putative oncogene are not well defined. DEK transcription was previously shown to be induced by the high-risk human papillomavirus (HPV) E7 oncogene via E2F and Rb pathways. Transient DEK overexpression was able to inhibit both senescence and apoptosis in cultured cells. In at least the latter case, this mechanism involved the destabilization of p53 and the decreased expression of p53 target genes. We show here that DEK overexpression disrupts the normal differentiation program in a manner that is independent of either p53 or cell death. DEK expression was distinctly repressed upon the differentiation of cultured primary human keratinocytes, and stable DEK overexpression caused epidermal thickening in an organotypic raft model system. The observed hyperplasia involved a delay in keratinocyte differentiation toward a more undifferentiated state, and expansion of the basal cell compartment was due to increased proliferation, but not apoptosis. These phenotypes were accompanied by elevated p63 expression in the absence of p53 destabilization. In further support of bona fide oncogenic DEK activities, we report here up-regulated DEK protein levels in both human papilloma virus-positive hyperplastic murine skin and a subset of human squamous cell carcinomas. We suggest that DEK up-regulation may contribute to carcinoma development at least in part through increased proliferation and retardation of differentiation. The human DEK protein was originally identified as a fusion with the NUP214/CAN nucleoporin in a subset of patients with acute myeloid leukemia 1 and was independently purified as a protein that modulates the topology of SV40 minichromosomes.2 DEK is abundantly expressed in proliferating cells, and a majority of the protein is bound to chromatin, whereas a small fraction is bound to RNA.3 The 43-kDa nuclear phosphoprotein is the only member of its family, and contains a conserved central SAP DNA binding domain with homology to SAF-A/B, acinus, and PIAS, 4 and a second DNA binding motif within the C-terminus.5,6 DNA binding as well as DEK self-association can be regulated by C-terminal phosphorylation and N-terminal acetylation. 7,8 Preferential DEK association with structured DNA templates and its ability to induce positive supercoils into circular DNA templates in vitro have led to the notion that DEK serves as an architectural protein.9 -12 Multiple reports have implicated DEK in replication, 2 positive and negative regulation of transcription [13][14][15][16][17][18][19] as well as mRNA processing. 20 -22 How these activities translate into putative oncogenic DEK functions is presently unclear.

k High-risk human papillomaviruses (HPVs) deregulate epidermal differentiation and cause anogenital and head and neck squamous cell carcinomas (SCCs). The E7 gene is considered the predominant viral oncogene and drives proliferation and genome instability. While the implementation of routine screens has greatly reduced the incidence of cervical cancers which are almost exclusively HPV positive, the proportion of HPV-positive head and neck SCCs is on the rise. High levels of HPV oncogene expression and genome load are linked to disease progression, but genetic risk factors that regulate oncogene abundance and/or genome amplification remain poorly understood. Fanconi anemia (FA) is a genome instability syndrome characterized at least in part by extreme susceptibility to SCCs. FA results from mutations in one of 15 genes in the FA pathway, whose protein products assemble in the nucleus and play important roles in DNA damage repair. We report here that loss of FA pathway components FANCA and FANCD2 stimulates E7 protein accumulation in human keratinocytes and causes increased epithelial proliferation and basal cell layer expansion in the HPV-positive epidermis. Additionally, FANCD2 loss stimulates HPV genome amplification in differentiating cells, demonstrating that the intact FA pathway functions to restrict the HPV life cycle. These findings raise the possibility that FA genes suppress HPV infection and disease and suggest possible mechanism(s) for reported associations of HPV with an FA cohort in Brazil and for allelic variation of FA genes with HPV persistence in the general population.H uman papillomaviruses (HPVs) are double-stranded DNA viruses comprised of more than 100 subtypes, some of which, designated high risk, cause cervical and approximately one quarter of head and neck squamous cell carcinomas (HNSCCs) (1,20,21). HPV is associated primarily with HNSCCs that occur in the oropharynx, which includes the back of the tongue, walls of the throat, the soft palate, and the tonsils. Approximately 60% of oropharyngeal SCCs are caused by one of the high-risk types of HPV, and the vast majority of those are positive for HPV type 16 (HPV16) (10). Importantly, these HNSCCs are a distinct type distinguishable from HPV-negative tumors which have alcohol and tobacco use as the predominant risk factors (1).HPV is the causative agent of nearly all cervical cancers and is best studied in that disease. Although HPV infections are common, with up to 90% of women estimated to be infected in their lifetime, most infections are cleared by the immune system and do not progress to cancer. A small fraction of HPV infections, however, lead to low-grade and high-grade squamous intraepithelial lesions and cervical cancer. Persistent infection is the most significant risk factor for developing cervical cancer. While the exact progression of events is still not entirely understood, replication, persistence, integration of the viral genome, and increased expression of the viral E6 and E7 oncogenes are important steps in malignant pro...