DNA Polymerase Clamp Shows Little Turnover at Established Replication Sites but Sequential De Novo Assembly at Adjacent Origin Clusters

Abstract

The spatial and temporal organization of DNA replication was investigated in living cells with a green fluorescent protein fusion to the DNA polymerase clamp PCNA. In situ extractions and photobleaching experiments revealed that PCNA, unlike RPA34, shows little if any turnover at replication sites, suggesting that it remains associated with the replication machinery through multiple rounds of Okazaki fragment synthesis. Photobleaching analyses further showed that the transition from earlier to later replicons occurs by disassembly into a nucleoplasmic pool of rapidly diffusing subcomponents and reassembly at newly activated sites. The fact that these replication sites were de novo assembled in close proximity to earlier ones suggests that activation of neighboring origins may occur by a domino effect possibly involving local changes in chromatin structure and accessibility.

Cell cycle specific distribution of killin: evidence for negative regulation of both DNA and RNA synthesis

“…Thereafter, a cascade of events occur, including the loading of the ring-shaped replication factor PCNA that ensure processivity of DNA replication. Both RFP-labeled RPA and GFP-PCNA have been previously colocalized with active replication foci (containing multiple replication forks) visualized by BrdU labeling, 22,29 as well as their largely co-localization pattern in S-phase nuclei shown in this study.…”

“…To this end, we conducted the salt extraction which has been shown to disrupt GFP-PCNA signal in non-S phase nuclei, while GFP-PCNA signal associated with DNA replication forks during S phase remained unperturbed. 29 When Cos-E5 cells double transfected with GFP-PCNA and RFP control were treated with 300 mM NaCl, we noticed that none of the S-phase cells marked by punctate nuclear GFP-PCNA foci (replication forks) had any RFP signal compared to untreated cells shown in Fig. 1B (Fig.…”

“…Since pTEN functions as negative regulator of AKT while Killin in p53-mediated S-phase arrest, the extreme close proximity of the 2 genes share by a divergent promoter makes pTEN/killin locus a potent tumor-suppressor dual, and the loss of function in either gene seems to predispose one to increased risk of cancer, as seen in patients with Cowden syndrome. 18,[30][31][32][33] Another interesting finding of our study is the revelation that in non-S-phase cells marked by diffusive expression of GFP-PCNA, 22,29 we notice RFP-Killin signals congregate in nucleolus. When these cells were treated with salt extraction prior to fluorescence microscopy, we show that GFP-PCNA signals, in contrast to those being at S-phase described above, are completely lost, whereas RFP-Killin signals remain in the nucleoli of the cells.…”

“…29 Briefly, RFP or RFPKillin were transiently co-transfected and with GFP-PCNA into Cos-E5 cells for 24 h. Cells grown were then permeabilized for about 30 seconds with ice-cold CSK buffer (50 mM NaCl, 250 mM sucrose, 2 mM MgCl 2 , 2 mM EGTA, 10 mM PIPES, pH 6.8) containing 0.1% Triton-X 100. Thereafter, the cells were further extracted for 1 min with ice-cold phosphate buffer containing 300 mM of NaCl.…”

Nucleosome assembly and epigenetic inheritance

“…Using high resolution imaging techniques, Taddei et al (1999) provided evidences that CAF-1 persists on newly replicated DNA after its synthesis for a period of more than 20 min, in consistence with the in vitro finding that CAF-1 assembles nucleosome post-replication (Shibahara and Stillman, 1999). In addition, a similar phenomenon was also observed on PCNA by fluorescent bleaching experiment (Sporbert et al, 2002). Hence, the association of PCNA and CAF-1 on replicated DNA could present a time window for synergistic restoration of chromatin structure and epigenetic marks.…”