POSTEPY BIOLOGII KOMORKI 36 (1): 55-76 2009
Rybaczek, D, Uniwersytetu Lodzkiego, Katedra Cytofizjol, Ul Pilarskiego 14, PL-90231 Lodz, Poland.
A wide array of research programs have been directed towards a comprehension of roles the intra-S-phase checkpoint which controls either frequency of DNA replication initiation (origin densities) or replication fork movement (rates of elongation). In response to treatment with either hydroxyurea or aphidicolin and after the addition of the DNA-damaging agents, the total rate of DNA replication per cell is reduced. This reduction is due to activation of an intra-S-phase checkpoint-dependent biochemical pathway network. The activated intra-S-phase checkpoint slows down or arrests replication forks, inhibits the premature firing of late origins, starts Lip the DNA damage response pathways to prevent replication of a damaged DNA, and delays the onset of mitosis until the cells are exposed to replicational stress. This work focuses on ATR, ATM, Chk1 and Chk2 protein kinases that are required for the control of the S phase, illustrates the state of knowledge about the other protei! ns involved in DNA-replication stress-response, and in addition explains their relationship. Ataxia telangiectasia mutated kinase (ATM) and ataxia telangiectasia and Rad3-related kinase (ATR) are PI-3 Kinase-related Kinase (PIKK) family members. Despite the essential role of ATM and ATR in cell cycle signaling, little is known about their activation. The activated ATM and ATR kinases turn on their downstream target proteins (like Chk2 and Chk1) by phosphorylating specific serine or threonine residues. ATM responds primarily to double strand breaks and phosphorylates Chk2 protein kinase at the amino-terminal domain contains a threonine residue (Thr68). Phosphorylation on Thr68 is a precondition for the successive activation step.. which is attributable to autophosphorylation of Chk2 on Thr383 and Thr387. ATR is activated by replicational stress or UV-induced DNA damages and in response phosphorylates Chk1 protein kinase at serine residues (Ser317 and Ser345). Phosphorylation! at Ser345 serves to localize Chk1 to the nucleus following ch! eckpoint activation, while phosphorylation at Ser317 was shown to forbid entry into G2 phase and mitosis following stalled DNA replication. It is known, however, that ATM and ATR protein kinases share some phosphorylation targets and their precise roles in the intra-S-phase checkpoint pathway may differ depending on the nature of stress involved. Chk1-mediated Cdc25A-C phosphorylation leading to blocking of CdkI and Cdk2 (thus preventing cell cycle progression). Chk1 can stabilize the replisome, possibly by targeting replication proteins (e.g., Cdc6, MCM2-7), and after resolving the replication problems can restart of stalled replication forks. Functional changeability of the ATM/ATR-Chk2/Chk1-Cdc25/Cdk axis underlie the molecular foundation of the intra-S-phase checkpoint. ATR also phosphorylates histone H2AX on serine 139. After DSB-like DNA damage a number of Ser139-phosphorylated-H2AX localizes to sites of DNA damage at subnuclear foci. Although most of them spread throughout th! e whole area of nucleoplasm, the largest of them, localized at perinucleolar heterochromatin regions. This newly phosphorylated-H2AX forming a platform for the recruitment DNA repair and signaling proteins. This paper also briefly describes abrogating the intra-S-phase checkpoint function will result in overriding the S-M dependency and induction of premature chromosome condensation (PCC). Apart from numerous mutations that eliminate particular elements of the intra-S-phase checkpoint pathway, systems which monitor the course of DNA replication can be affectd by many types of chemical agents. Caffeine, can override the S-M dependency and induce PCC in cells not prepared to undertake mitotic division, i.e. those which did not complete DNA replication and stay underreplicated. S-phase-blocked cells treated with caffeine start Out aberrant mitotic divisions. The full array of aberrations includes: chromosomal breaks and gaps lost and lagging chromatids and chromosomes, chromosome bridges and micronuclei. Thus, drug-induced PCC (due to caffeine action) clearly provided the new insight that DNA replication is tightly coupled with the construction of the higher-ordered structure of the eukaryote chromosome. In the hope of unraveling targets for cytostatic drugs and cellular factors which inhibit or potentiate healing of cancer, a wide array of research programs have! been directed towards an understanding of molecular mechanisms that underlie the intra-S-phase signaling pathways. A bulk of research-work is thus focused on methods increasing the effects of radio- and chemotherapy.