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The migration of chromosomes during mitosis is mediated primarily by kinesins that bind to the chromosomes and move along the microtubules, exerting pulling and pushing forces on the centrosomes. We report that a DNA replication protein, Sld5, localizes to the centrosomes, resisting the microtubular pulling forces experienced during chromosome congression. In the absence of Sld5, centriolar satellites, which normally cluster around the centrosomes, are dissipated throughout the cytoplasm, resulting in the loss of their known function of recruiting the centrosomal protein, pericentrin. We observed that Sld5-deficient centrosomes lacking pericentrin were unable to endure the CENP-E- and Kid-mediated microtubular forces that converge on the centrosomes during chromosome congression, resulting in monocentriolar and acentriolar spindle poles. The minus-end-directed kinesin-14 motor protein, HSET, sustains the traction forces that mediate centrosomal fragmentation in Sld5-depleted cells. Thus, we report that a DNA replication protein has an as yet unknown function of ensuring spindle pole resistance to traction forces exerted during chromosome congression.

The primary eukaryotic single-stranded DNA-binding protein, Replication protein A (RPA), binds to single-stranded DNA at the sites of DNA damage and recruits the apical checkpoint kinase, ATR via its partner protein, ATRIP. It has been demonstrated that absence of RPA incapacitates the ATR-mediated checkpoint response. We report that in the absence of RPA, human single-stranded DNA-binding protein 1 (hSSB1) and its partner protein INTS3 form sub-nuclear foci, associate with the ATR-ATRIP complex and recruit it to the sites of genomic stress. The ATRIP foci formed after RPA depletion are abrogated in the absence of INTS3, establishing that hSSB-INTS3 complex recruits the ATR-ATRIP checkpoint complex to the sites of genomic stress. Depletion of homologs hSSB1/2 and INTS3 in RPA-deficient cells attenuates Chk1 phosphorylation, indicating that the cells are debilitated in responding to stress. We have identified that TopBP1 and the Rad9-Rad1-Hus1 complex are essential for the alternate mode of ATR activation. In summation, we report that the single-stranded DNA-binding protein complex, hSSB1/2-INTS3 can recruit the checkpoint complex to initiate ATR signaling.

When mammalian cells experience DNA damaging stress, they block DNA replication to avoid erroneous replication of the damaged template. The cells that are unable to respond to DNA damage continue faulty DNA replication that results in incorporation of genomic lesions. To understand the regulation of replication machinery during stress, systemic studies have been carried out but they have been restricted to the evaluation of the mRNA levels and therefore have not been able to identify post-transcriptional changes, vital for immediate blocking of the progressing DNA replication. We have recently discovered that an essential replication factor is downregulated by radiation stress. In this study, we have carried out a systematic evaluation of protein levels of entire replication apparatus after different types of DNA damage. We report that, independent of the status of p53 and retinoblastoma protein, mammalian cells choose targets that are essential for prereplication, preinitiation, and elongation phases of replication. We imposed different kinds of stress to discern whether similar or unique responses are invoked, and we propose a model for inhibition of replication machinery in which mammalian cells target specific essential replication factors based on the experienced stress. © 2010 IUBMB IUBMB Life, 62(10): 764–775, 2010

Background Mcm10 protein is essential for initiation and elongation phases of replication. Human cells proteolyze Mcm10 during mitosis, presumably to ensure a single round of replication. It has been proposed that anaphase promoting complex ubiquitinates Mcm10 in late M and early G1 phases. Results In contrast to the previous work, we report that the degradation of Mcm10 is initiated at the onset of mitosis. Immunoblotting and immunofluorescence assays display that Mcm10 levels are low in all phases of mitosis. We report that Mcm10 degradation is not dependent on anaphase promoting complex. Further, the proteolysis in M-phase can be independently mediated by non-overlapping regions of Mcm10, apparently employing a redundant mechanism to ensure downregulation. Conclusions It is believed that the proteolysis of Mcm10 during mitosis is a vital mechanism to prevent aberrant initiation of replication and the present study describes the regulation of Mcm10 during this phase of the cell-cycle.

We report that upon UV radiation insult, mammalian cells specifically down-regulate Mcm10, a protein essential for the initiation and elongation phases of DNA replication. The levels of a majority of replication factors remain unaffected under this condition, implying that Mcm10 is a key node in the regulation of the replication machinery. High doses of ionizing gamma radiation and exposure to a combination of DNA-damaging chemicals do not decrease Mcm10 protein levels, demonstrating that Mcm10 down-regulation is triggered only by UV-specific damage. The decrease of Mcm10 protein levels is not caused by transcriptional inhibition or cleavage by apoptotic enzymes, but results from degradation by the 26 S proteasome. UV-triggered degradation of Mcm10 requires its linker or C-terminal domain. In addition, Mcm10 down-regulation is not limited to cells from a particular lineage. Therefore, our study reveals a mechanism by which mammalian cells effectively inhibit the replication machinery during stress to prevent it from drifting toward a catastrophic path of genomic instability.