HUMAN REPLICATION PROTEINS HCDC21, HCDC46 AND P1MCM3 BIND CHROMATIN UNIFORMLY BEFORE S-PHASE AND ARE DISPLACED LOCALLY DURING DNA-REPLICATION

Members of the Mcm-protein family have recently been shown to be invol ved in restricting DNA replication to a single cycle in Xenopus laevis egg extracts. In this study, we extended these observations to human somatic cells and analysed the localisation of the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 in replicating HeLa cell nuclei. These Mcm-pro teins are entirely nuclear in interphase cells and apparently exist in two populations: a nucleosolic population, and a population bound to a nuclear structure, most likely chromatin. The bound population is de tected throughout the nucleus in late G(1) and early S, and ale discre te subnuclear sites following further progression of S-phase. We use h igh resolution confocal microscopy to determine the subnuclear sites o f chromatin-bound Mcm proteins in comparison to the sites of replicati ng DNA. Importantly, hCdc21, hCdc46 and P1Mcm3 do not co-localise with replication foci, instead these proteins appear to coincide with subn uclear sites of unreplicated chromatin. During progression of S-phase hCdc21, hCdc46 and P1Mcm3 are displaced from their site on chromatin a t the time when this site is replicated. Consequently, early replicati ng sites do not contain bound hCdc21, hCdc46 or P1Mcm3 during later st ages of S-phase. Furthermore, G(2) nuclei and condensed chromatin in m itotic cells do not contain bound hCdc21, hCdc46 or P1Mcm3. Thus, the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 are not concentrated at sit es of DNA replication. Instead, they appear to be present only on unre plicated chromatin and are displaced from replicating chromatin, consi stent with a role in monitoring unreplicated chromatin and ensuring on ly a single round of DNA replication per cell cycle.