X-shaped Structures Research Articles

Rec'd and repaired

DNA replication stalls when the polymerase encounters lesions in the DNA, but recovers soon after lesion repair. In a recent work, Justin Courcelle and colleagues (Mississippi State University, Mississippi State, MS) examine what happens to the replication fork during this downtime. The results show that maintaining the correct fork structure depends on recombination proteins that may help to prevent illegitimate strand exchanges. Figure The structures of replicating DNAs change (left to right) to allow repair work. Courcelle's group used two-dimensional gel electrophoresis to examine the shapes of a replicating bacterial plasmid. Advancing replication forks yielded the expected Y-shaped structure. But UV-induced lesions stalled the replication fork and produced X-shaped structures. These structures represent the nascent DNA backing up from the apex of a Y-shaped fork. The stalled structures were processed by RecQ and RecJ and maintained by RecA and RecF, which are the same proteins that promote homologous DNA pairing during recombinational processes. A mid-replication stall is “like catching a cell with its pants down,” according to Courcelle. “It can't live as one and a half cells for eternity,” he says. Unchecked free DNA ends are recombinogenic. Fork stabilization by these Rec proteins may be essential for preventing unwanted mitotic recombination and its potentially cancerous consequences. In addition, fork regression and Rec binding probably delays replication long enough for repair enzymes or SOS polymerases either to repair the lesion or to replicate past it. Whether RecA and RecF recruit repair enzymes or simply maintain an open fork remains to be determined. ▪ Reference: Courcelle, J., et al. 2003. Science. 10.1126/science.1081328.

Mus81-Eme1 Are Essential Components of a Holliday Junction Resolvase

Mus81, a fission yeast protein related to the XPF subunit of ERCC1-XPF nucleotide excision repair endonuclease, is essential for meiosis and important for coping with stalled replication forks. These processes require resolution of X-shaped DNA structures known as Holliday junctions. We report that Mus81 and an associated protein Eme1 are components of an endonuclease that resolves Holliday junctions into linear duplex products. Mus81 and Eme1 are required during meiosis at a late step of meiotic recombination. The mus81 meiotic defect is rescued by expression of a bacterial Holliday junction resolvase. These findings constitute strong evidence that Mus81 and Eme1 are subunits of a nuclear Holliday junction resolvase.

The structure of 4-way DNA junctions: specific binding of bis-intercalators with rigid linkers.

During replication and recombination, two DNA duplexes lie side by side. We have developed reagents that might be used to probe structure during these critical processes; they contain two intercalating groups connected by a rigid linker that forces those groups to point in opposite directions. If their stereochemistry proves appropriate, such structure-specific agents should intercalate specifically into adjacent duplexes in the Y- and X-shaped structures (i.e. 3- and 4-way junctions, now known as 3H and 4H junctions) found at replication and recombination sites. We prepared DNA structures in which four duplexes were arranged in all possible combinations around 2- and 4-way junctions and then probed the accessibility to DNase I of all their phosphodiester bonds. In the absence of any bis-intercalators, 7-9 nucleotides (nt) in each of the strands in 4-way junctions were protected from attack; protected regions were significantly offset to the 3' side of the junction in continuous strands, but only slightly offset, if at all, in exchanging strands. All the intercalators decreased accessibility throughout the structure, but none did so at specific points in the two adjacent arms of 4-way junctions. However, one bis-intercalator--but not its sister with a shorter linker--strikingly increased access to a particular CpT bond that lay 9 nt away from the centre of some 4-way junctions without reducing access to neighbouring bonds. Binding was both sequence and structure specific, and depended on complementary stereochemistry between bis-intercalator and junction.