Repair of dsDNA breaks requires processing to produce 39-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); and Replication protein A (RPA). Resection occurs via two routes. In one, BLM and DNA2 physically and specifically interact to resect DNA in a process that is ATP-dependent and requires BLM helicase and DNA2 nuclease functions. RPA is essential for both DNA unwinding by BLM and enforcing 59 / 39 resection polarity by DNA2. MRN accelerates processing by recruiting BLM to the end. In the other, EXO1 resects the DNA and is stimulated by BLM, MRN, and RPA. BLM increases the affinity of EXO1 for ends, and MRN recruits and enhances the processivity of EXO1. Our results establish two of the core machineries that initiate recombinational DNA repair in human cells.

Breaks in both DNA strands are a particularly dangerous threat to genome stability. At a DNA double-strand break (DSB), potentially lost sequence information cannot be recovered from the same DNA molecule. However, simple repair by joining two broken ends, though inherently error prone, is preferable to leaving ends broken and capable of causing genome rearrangements. To avoid DSB-induced genetic disinformation and disruption of vital processes, such as replication and transcription, cells possess robust mechanisms to repair DSBs. Because all breaks are not created equal, the particular repair mechanism used depends largely on what is possible and needed based on the structure of the broken DNA. We argue that although categorizing different DSB repair mechanisms along pathways and subpathways can be conceptually useful, in cells flexible and reversible interactions among DSB repair factors form a web from which a nonpredetermined path to repair for any number of different DNA breaks will emerge.

SUMMARY MRE11 within the MRE11-RAD50-NBS1 (MRN) complex acts in DNA double-strand break repair (DSBR), detection and signaling; yet, how its endo- and exonuclease activities regulate DSB repair by non-homologous end-joining (NHEJ) versus homologous recombination (HR) remains enigmatic. Here we employed structure-based design with a focused chemical library to discover specific MRE11 endo- or exonuclease inhibitors. With these inhibitors we examined repair pathway choice at DSBs generated in G2 following radiation exposure. Whilst endo- or exonuclease inhibition impairs radiation-induced RPA chromatin binding, suggesting diminished resection, the inhibitors surprisingly direct different repair outcomes. Endonuclease inhibition promotes NHEJ in lieu of HR, whilst exonuclease inhibition confers a repair defect. Collectively, the results describe nuclease-specific MRE11 inhibitors, define distinct nuclease roles in DSB repair, and support a mechanism whereby MRE11 endonuclease initiates resection, thereby licensing HR followed by MRE11 exo and EXO1/BLM bidirectional resection towards and away from the DNA end, which commits to HR.

The human Rad50 protein, classified as a structural maintenance of chromosomes (SMC) family member, is complexed with Mre11 (R/M) and has important functions in at least two distinct double-strand break repair pathways. To find out what the common function of R/M in these pathways might be, we investigated its architecture. Scanning force microscopy showed that the complex architecture is distinct from the described SMC family members. R/M consisted of two highly flexible intramolecular coiled coils emanating from a central globular DNA binding domain. DNA end-bound R/M oligomers could tether linear DNA molecules. These observations suggest that a unified role for R/M in multiple aspects of DNA repair and chromosome metabolism is to provide a flexible, possibly dynamic, link between DNA ends.

Repair of interstrand crosslinks (ICLs) requires multiplestrand incisions to separate the two covalently attached strands of DNA. It is unclear how these incisions are generated. DNA double-strand breaks (DSBs) have been identified as intermediates in ICL repair, but enzymes responsible for producing these intermediates are unknown. Here we show that Mus81, a component of the Mus81-Eme1 structure-specific endonuclease, is involved in generating the ICL-induced DSBs in mouse embryonic stem (ES) cells in S phase. Given the DNA junction cleavage specificity of Mus81-Eme1 in vitro, DNA damage-stalled replication forks are suitable in vivo substrates. Interestingly, generation of DSBs from replication forks stalled due to DNA damage that affects only one of the two DNA strands did not require Mus81. Furthermore, in addition to a physical interaction between Mus81 and the homologous recombination protein Rad54, we show that Mus81 À/À Rad54 À/À ES cells were as hypersensitive to ICL agents as Mus81 À/À cells. We propose that Mus81-Eme1-and Rad54-mediated homologous recombination are involved in the same DNA replication-dependent ICL repair pathway.

Cytoplasmic linker protein (CLIP)-170, CLIP-115, and the dynactin subunit p150Glued are structurally related proteins, which associate specifically with the ends of growing microtubules (MTs). Here, we show that down-regulation of CLIP-170 by RNA interference results in a strongly reduced accumulation of dynactin at the MT tips. The NH2 terminus of p150Glued binds directly to the COOH terminus of CLIP-170 through its second metal-binding motif. p150Glued and LIS1, a dynein-associating protein, compete for the interaction with the CLIP-170 COOH terminus, suggesting that LIS1 can act to release dynactin from the MT tips. We also show that the NH2-terminal part of CLIP-170 itself associates with the CLIP-170 COOH terminus through its first metal-binding motif. By using scanning force microscopy and fluorescence resonance energy transfer-based experiments we provide evidence for an intramolecular interaction between the NH2 and COOH termini of CLIP-170. This interaction interferes with the binding of the CLIP-170 to MTs. We propose that conformational changes in CLIP-170 are important for binding to dynactin, LIS1, and the MT tips.

Nitrogen regulatory protein C (NtrC) contacts a bacterial RNA polymerase from distant enhancers by means of DNA loops and activates transcription by allowing polymerase to gain access to the template DNA strand. It was shown that NtrC from Salmonella typhimurium must build large oligomers to activate transcription. In contrast to eukaryotic enhancer-binding proteins, most of which must bind directly to DNA, some NtrC dimers were bound solely by protein-protein interactions. NtrC oligomers were visualized with scanning force microscopy. Evidence of their functional importance was provided by showing that some inactive non-DNA-binding and DNA-binding mutant forms of NtrC can cooperate to activate transcription.

The Escherichia coli H-NS protein is a nucleoid-associated protein involved in gene regulation and DNA compaction. To get more insight into the mechanism of DNA compaction we applied atomic force microscopy (AFM) to study the structure of H-NS-DNA complexes. On circular DNA molecules two different levels of H-NS induced condensation were observed. H-NS induced lateral condensation of large regions of the plasmid. In addition, large globular structures were identified that incorporated a considerable amount of DNA. The formation of these globular structures appeared not to be dependent on any specific sequence. On the basis of the AFM images, a model for global condensation of the chromosomal DNA by H-NS is proposed.