Initiation at the human Fragile X (FMR1) origin of chromosomal replication

Abstract

Eukaryotic chromosomal DNA replication begins at origins marked by pre‐replication complexes (pre‐RCs) containing ORC, MCM2‐7, and other proteins. However, understanding of mammalian origins and their possible role in maintenance of genomic stability remains incomplete. We have mapped a replication origin 5' of the FMR1 gene, a heritable fragile site on the human X chromosome. As expected, the FMR1 origin binds to pre‐RC proteins in vivo. FMR1 alleles with heritable CGG repeat expansions suffer chromosome breaks and gaps when cells experience folate deficiency, but how the expansion leads to chromosome fragility is not clear.Recent clues from the human DNA helicase HDHB may lead to one answer for this question. In late M/G1, HDHB assembles in pre‐RCs at the laminB2, MCM4, and FMR1 origins, but not in flanking sequences. Based on siRNA and dominant negative approaches, HDHB is important for initiation. At G1/S, HDHB is released from the laminB2 and MCM4 origins and much of it is exported from the nucleus. However, a low level of HDHB is retained on bulk chromatin during S phase and this fraction is increased in cells exposed to agents that stall fork progression. To ask whether HDHB may localize at stalled replication forks even in unperturbed cells, we used ChIP to study the FMR1 origin region and the common fragile site FRA16D. HDHB was strongly enriched in S phase at both sites and not in flanking regions. Taken together, the data suggest that HDHB functions in initiation of replication at origins and in preventing or repairing stalled replication forks that arise in specific regions even in unperturbed cells.