Decatenation G2 Checkpoint

 

DNA topoisomerases are evolutionarily conserved nuclear enzymes that have major functions in the cell cycle including DNA replication, recombination, and chromosome segregation. The two major families of topoisomerases (type I and type II) are differentiated by the type of enzymatic reaction each one performs. Type I topoisomerases produce protein-associated single-strand breaks (ssb) in DNA and relieve supercoiling tension by free rotation of the cut strand around the intact strand. Type II topoisomerases produce protein-associated DNA double-strand breaks (dsb) and are capable of passing an intact DNA duplex through the protein-associated dsb. Thus, only type II topoisomerases can separate knotted and intertwined DNA molecules. 
 
Mammalian species express two type II topoisomerase isoforms: a and β. Each isoform is encoded by a separate gene located in different human chromosomes (chromosomes 17 and 3, respectively) and can be distinguished by mass (~170 and ~180 kDa, respectively). The two isoforms share ~70 % homology at the amino acid level, and both isoforms form a homodimer enzymatic complex with similar catalytic activity. In addition, α/β heterodimers have been detected in HeLa cells

 

In contrast to topo IIβ, which is non-essential and constitutively expressed, topo IIa is an essential gene and is expressed at its greatest levels in G2 and M. It is thought that topo II activity is required for chromosome condensation, decatenation of intertwined and knotted daughter DNA duplexes prior to anaphase, and centromere resolution. Catenations between chromatid arms appear to be removed prior to mitosis while centromeric catenations persist up to the metaphase/anaphase transition. In addition to its role in chromosome segregation, topo IIa is abundant in the nuclear matrix and the chromosome scaffold where it may help to attach chromatin loops. 

DNA and topo IIa form a reversible, covalent DNA-topoisomerase complex, often referred to as the cleavage complex. Under physiological conditions, this transient intermediary complex has a very short half-life (e.g. milliseconds to a second). Inhibitory drugs interfere with various stages of the catalytic cycle, and therefore topo II inhibitors are divided into two classes: poisons and catalytic inhibitors. Topo II poisons such as doxorubicin (Adriamycin) and etoposide (VP-16) stabilize the cleavage complex for minutes to hours. The stable DNA-topo II cleavage complex may collide with DNA replication forks or transcriptional machinery and create chromatid breaks. Upon proteolysis of the stabilized cleaved complex, a DNA dsb is exposed, similar to the effects of ionizing radiation (IR) treatment. Thus, these drugs are used clinically to treat cancers which typically overexpress type II topoisomerases.
 
Unlike topo II poisons, catalytic inhibitors prevent formation of the cleavage complex by several different mechanisms. For example, aclarubicin inhibits DNA binding to topo II by intercalating into DNA, whereas ICRF-193 stabilizes the topo II in a closed-clamp conformation after the ligation step of the catalytic cycle. Although, the topo II poisons have been widely used as potent anti-cancer drugs, topo II catalytic inhibitors are primarily used in the clinical setting as an adjunct to reduce the cardiotoxicity of topo II poisons. The ability of topo II catalytic inhibitors such as ICRF-187 and ICRF-193 to protect against the toxicity of topo II poisons implies that poisons and catalytic inhibitors have very different effects on topo II activity, DNA, and chromatin.
 
Studies using topo II catalytic inhibitors such as ICRF-193 suggest that G2 cells monitor the state of decatenation of intertwined sister chromatids following DNA replication and actively delay progression into mitosis pending sufficient chromatin decatenation.  A subset of chromatid catenations appear to be organized in the centromere, and these catenations are not separated until the onset of anaphase.  Thus, the decatenation G2 checkpoint appears to monitor the sufficiency of decatenation, not its completion. The expression of an active decatenation G2 checkpoint was supported by observations showing that the G2 delay in cells with catalytic inhibition of topo II was an active process that was dependent upon BRCA1 expression and could be overridden by the use of general kinase inhibitors such as caffeine. BRCA1 may be required to activate topo IIa by ubiquitylation as well as mediate G2 delay. Other studies have implicated Plk1, WRN, and Chk1 in the decatenation G2 checkpoint. Although the presence of a decatenation G2 checkpoint that is independent of DNA damage has been supported by a variety of studies, the concept is still controversial. For example, ICRF-193 has been shown to induce DNA damage and activate  DNA damage signaling in some cancer cell lines, and therefore it has been difficult to distinguish between the DNA damage and decatenation G2 checkpoints.
 
We have recently shown that normal human diploid fibroblast (NHDF) lines isolated from three different individuals and depleted of topo IIα display a loss of decatenation G2 checkpoint function and abnormal allele frequencies of the p16INK4A tumor suppressor, suggesting that topo IIα is required to maintain genomic stability. The data also supported a distinction between the decatenation and DNA damage G2 checkpoints. Furthermore, depletion of topo IIα bypassed activation of the decatenation G2 checkpoint, allowed mitotic entry in the presence of catenated chromatids, caused chromosomal mis-segregation during mitosis, and resulted in genomic instability.  Our current work is focused on defining the signaling mechanisms involved in the decatenation G2 checkpoint and thus, the maintenance of genomic stability.