In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment. Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A. Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate α-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome. A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg 80 and Gly 81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans-acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.

SummaryRAD52 mediates homologous recombination by annealing cDNA strands. However, the detailed mechanism of DNA annealing promoted by RAD52 has remained elusive. Here we report two crystal structures of human RAD52 single-stranded DNA (ssDNA) complexes that probably represent key reaction intermediates of RAD52-mediated DNA annealing. The first structure revealed a “wrapped” conformation of ssDNA around the homo-oligomeric RAD52 ring, in which the edges of the bases involved in base pairing are exposed to the solvent. The ssDNA conformation is close to B-form and appears capable of engaging in Watson-Crick base pairing with the cDNA strand. The second structure revealed a “trapped” conformation of ssDNA between two RAD52 rings. This conformation is stabilized by a different RAD52 DNA binding site, which promotes the accumulation of multiple RAD52 rings on ssDNA and the aggregation of ssDNA. These structures provide a structural framework for understanding the mechanism of RAD52-mediated DNA annealing.

Rad52 plays essential roles in homology-dependent doublestrand break repair. Various studies have established the functions of Rad52 in Rad51-dependent and Rad51-independent repair processes. However, the precise molecular mechanisms of Rad52 in these processes remain unknown. In the present study we have identified a novel DNA binding site within Rad52 by a structure-based alanine scan mutagenesis. This site is closely aligned with the putative single-stranded DNA binding site determined previously. Mutations in this site impaired the ability of the Rad52-single-stranded DNA complex to form a ternary complex with double-stranded DNA and subsequently catalyze the formation of D-loops. We found that Rad52 introduces positive supercoils into double-stranded DNA and that the second DNA binding site is essential for this activity. Our findings suggest that Rad52 aligns two recombining DNA molecules within the first and second DNA binding sites to stimulate the homology search and strand invasion processes.The repair of DSBs 4 is a critical process by which cells maintain genome integrity. In eukaryotic cells these breaks are repaired by homologous recombination (HR) and non-homologous end joining. In the homologous end joining pathway, broken DNA ends are rejoined by religation or annealing of short common sequences (microhomologies) near their ends.Because homologous end joining lacks a mechanism to reject erroneous pairings that occur by chance, recessed DNA, chromosome rearrangements, and mutations are possible outcomes of this repair pathway (1). By contrast, the HR pathway achieves high accuracy in DSB repair by utilizing mechanisms for a DNA homology search. In this pathway the ends of the DSBs are resected to generate 3Ј single-stranded tails. The singlestranded region then either invades an undamaged homologous duplex DNA (strand invasion) or pairs with a complementary single strand (single-strand annealing). The importance of this pathway is underscored by the high conservation, from yeast to humans, of the factors that catalyze HR (2).Rad52 is one of the key players in the yeast HR pathway, and its homologs have been found in higher eukaryotes. In yeast, Rad52 is essential in both the RAD51-dependent and -independent HR pathways and functions in several homology-dependent DSB repair events, including gene conversion, breakinduced replication, and recombination between inverted repeats (3). Several biochemical studies have established that Rad52 functions as a mediator in RAD51-dependent HR pathways. These findings include facilitating the assembly of the Rad51 recombinase on replication protein A-coated ssDNA (4, 5) and stimulating the DNA strand exchange activity of the Rad51 recombinase (6 -9). In vitro studies have also suggested more direct roles of Rad52 in HR. Rad52 catalyzes the annealing of complementary ssDNA and promotes D-loop formation and DNA strand exchange (10 -14). The strand invasion promoted by Rad52 may be relevant to some RAD51-independent HR events such as break-induced replication, in...

ObjectivesTo assess whether bipolar transurethral resection of the prostate (B-TURP) using the TURis ® system has a similar level of efficacy and safety to that of the traditional monopolar transurethral resection of the prostate (M-TURP), and to evaluate the impact of the TURis system on postoperative urethral stricture rates over a 36-month follow-up period. Patients and MethodsA total of 136 patients with benign prostatic obstruction were randomised to undergo either B-TURP using the TURis system or conventional M-TURP, and were regularly followed for 36 months after surgery. The primary endpoint was safety, which included the long-term complication rates of postoperative urethral stricture. The secondary endpoint was the follow-up measurement of efficacy. ResultsIn peri-operative findings, no patient in either treatment group presented with transurethral resection syndrome, and the decline in levels of haemoglobin and hematocrit were similar. The mean operation time was significantly extended in the TURis treatment group compared with the M-TURP group (79.5 vs 68.6 min; P = 0.032) and postoperative clot retention was more likely to be seen after M-TURP (P = 0.044). Similar efficacy findings were maintained throughout 36 months, but a significant difference in postoperative urethral stricture rates between groups was detected (6.6% in M-TURP vs 19.0% in TURis; P = 0.022). After stratifying patients according to prostate volume, there was no significant difference between the two treatment groups with regard to urethral stricture rates in patients with a prostate volume ≤ 70 mL (3.8% in M-TURP vs 3.8% in TURis), but in the TURis group there was a significantly higher urethral stricture rate compared with the M-TURP group in patients with a prostate volume >70 mL (20% in TURis vs 2.2% in M-TURP; P = 0.012). Furthermore, the mean operation time for TURis was significantly longer than for M-TURP for the subgroup of patients with a prostate volume > 70 mL (99.6 vs 77.2 min; P = 0.011), but not for the subgroup of patients with a prostate volume ≤ 70 mL. ConclusionThe TURis system seems to be as efficacious and safe as conventional M-TURP except that there was a higher incidence of urethral stricture in patients with larger preoperative prostate volumes.