DNA damage checkpoint responses in the S phase of synchronized diploid human fibroblasts.

Paul D Chastain,Haitao Chu,Marila Cordeiro‐Stone,Yingchun C Zhou,Bruna P Brylawski,Shangbang Rao,William K Kaufmann,Joseph G Ibrahim

doi:10.1111/php.12361

Abstract

We investigated the hypothesis that the strength of the activation of the intra-S DNA damage checkpoint varies within the S phase. Synchronized diploid human fibroblasts were exposed to either 0 or 2.5 J m−2 UVC in early, mid- and late-S phase. The endpoints measured were the following: (1) radio-resistant DNA synthesis (RDS), (2) induction of Chk1 phosphorylation, (3) initiation of new replicons and (4) length of replication tracks synthesized after irradiation. RDS analysis showed that global DNA synthesis was inhibited by approximately the same extent (30 ± 12%), regardless of when during S phase the fibroblasts were exposed to UVC. Western blot analysis revealed that the UVC-induced phosphorylation of checkpoint kinase 1 (Chk1) on serine 345 was high in early and mid S but 10-fold lower in late S. DNA fiber immunostaining studies indicated that the replication fork displacement rate decreased in irradiated cells at the three time points examined; however, replicon initiation was inhibited strongly in early and mid S, but this response was attenuated in late S. These results suggest that the intra-S checkpoint activated by UVC-induced DNA damage is not as robust toward the end of S phase in its inhibition of the latest firing origins in human fibroblasts.

Highlights

Eukaryotic cells have evolved a complex network of molecular reactions that work in concert to decrease the genotoxic effects of DNA damage
Replication forks progress more slowly in cells exposed to a DNA damaging agent than sham-treated cells [5,6,7]. This reduced fork displacement rate is thought to be a combination of both passive and active mechanisms; some replication forks stall upon encountering template lesions, while the rate of progression of others is actively reduced by a yet undefined intra-S checkpoint-mediated signaling mechanism that depends upon Tipin (Timeless-interacting protein), Hus1, checkpoint kinase 1 (Chk1) and XRCC3 (X-ray repair complementing defective in Chinese hamster cells 3) [6,8,9,10,11,12]
Normal human fibroblasts immortalized by expression of the catalytic subunit of human telomerase (NHF-hTERT) become quiescent at confluence and re-enter the cell cycle upon replating at a lower density

Summary

Introduction

Eukaryotic cells have evolved a complex network of molecular reactions that work in concert to decrease the genotoxic effects of DNA damage. Replication forks progress more slowly in cells exposed to a DNA damaging agent than sham-treated cells [5,6,7] This reduced fork displacement rate is thought to be a combination of both passive and active mechanisms; some replication forks stall upon encountering template lesions (passive inhibition), while the rate of progression of others is actively reduced by a yet undefined intra-S checkpoint-mediated signaling mechanism that depends upon Tipin (Timeless-interacting protein), Hus, Chk and XRCC3 (X-ray repair complementing defective in Chinese hamster cells 3) [6,8,9,10,11,12]

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