Evidence for BLM and Topoisomerase IIIalpha interaction in genomic stability

Abstract

The genomic instability of persons with Bloom's syndrome (BS) features particularly an increased number of sister-chromatid exchanges (SCEs). The primary cause of the genomic instability is mutation at BLM, which encodes a DNA helicase of the RecQ family. BLM interacts with Topoisomerase IIIalpha (Topo IIIalpha), and both BLM and Topo IIIalpha localize to the nuclear organelles referred to as the promyelocytic leukemia protein (PML) nuclear bodies. In this study we show, by analysis of cells that express various deletion constructs of green fluorescent protein (GFP)-tagged BLM, that the first 133 amino acids of BLM are necessary and sufficient for interaction between Topo IIIalpha and BLM. The Topo IIIalpha-interaction domain of BLM is not required for BLM's localization to the PML nuclear bodies; in contrast, Topo IIIalpha is recruited to the PML nuclear bodies via its interaction with BLM. Expression of a full-length BLM (amino acids 1-1417) in BS cells can correct their high SCEs to normal levels, whereas expression of a BLM fragment that lacks the Topo IIIalpha interaction domain (amino acids 133-1417) results in intermediate SCE levels. The deficiency of amino acids 133-1417 in the reduction of SCEs was not explained by a defect in DNA helicase activity, because immunoprecipitated 133-1417 protein had 4-fold higher activity than GFP-BLM. The data implicate the BLM-Topo IIIalpha complex in the regulation of recombination in somatic cells.

RecQ helicases: Multiple structures for multiple functions?

“…Instead, the double mutant induces a cellular response reminiscent of that seen following DNA damage, including focal accumulation of ␥-H2AX (Eladad et al, 2005). Despite the above discussion, Hu et al, 2001 found that the region of BLM required for PML body localization was in the N-terminal domain between residues 133 and 237 (Hu et al, 2001).…”

“…One clear role for the N-and C-terminal domains is to direct interactions with heterologous proteins. For example, BLM binds directly to topoisomerase III␣ via residues in the first 212 (likely 1-133) amino acids of the N-terminal domain, and to the RAD51 recombinase via both the N-terminal 1-212 region and the final C-terminal 150 residues (Hu et al, 2001;Johnson et al, 2000;Wu et al, 2000). Interestingly, both of these interactions and their approximate location in the RecQ helicase polypeptide are conserved between BLM and its yeast ortholog, Sgs1.…”

The MCM and RecQ Helicase Families: Ancient Roles in DNA Replication and Genomic Stability Lead to Distinct Roles in Human Disease

“…When mutants are prepared that do not allow BLM localization to PML-NB, there is about a two-fold increase in sister chromatid exchange. These findings indicate that there is a need for BLM-SUMO interaction in order for BLM to localize to PML-NB, and that BLM activity, such as its accumulation at stalled replication forks, may be regulated by this specific localization [168].…”

“…In addition, BLM is SUMO-1 and SUMO-2 modified [167]. When mutants are prepared in which the SUMO-binding sequences are deleted, BLM cannot localize to PML-NB [168]. When mutants are prepared that do not allow BLM localization to PML-NB, there is about a two-fold increase in sister chromatid exchange.…”

“…A central player, H2AX, is phosphorylated when DSBs are induced, and this phosphorylation involves ATM, ATR and DNA-PK. Over one million bp are then marked by phosphorylated H2AX (γH2AX) on each side of the break [112,[168][169][170][171]. γH2AX recruits additional repair proteins to the damage site [172].…”