Summary The Mre11/Rad50/NBS1 complex (MRN) maintains genomic stability by bridging DNA ends and initiating DNA damage signaling through activation of the ATM kinase. Mre11 possesses DNA nuclease activities that are highly conserved in evolution, but play unknown roles in mammals. To define functions of Mre11 we engineered targeted mouse alleles which either abrogate nuclease activities or inactivate the entire MRN complex. Mre11 nuclease deficiency causes a striking array of phenotypes indistinguishable from absence of MRN, including early embryonic lethality and dramatic genomic instability. We identify a crucial role for the nuclease activities in homology directed double strand break repair, and a contributing role in activating the ATR kinase. However, nuclease activities are not required to activate ATM after DNA damage or telomere deprotection. Therefore, nucleolytic processing by Mre11 is an essential function of fundamental importance in DNA repair distinct from MRN control of ATM signaling.

Summary The Mre11/Rad50/NBS1 (MRN) complex plays many roles in response to DNA double strand breaks (DSBs), but its functions in repair by non homologous end joining (NHEJ) pathways are poorly understood. We have investigated requirements for MRN in Class Switch Recombination (CSR), a programmed DNA rearrangement in B lymphocytes that requires NHEJ. To this end we have engineered mice that lack the entire MRN complex in B lymphocytes, or possess an intact complex harboring mutant Mre11 lacking DNA nuclease activities. MRN deficiency confers a striking defect in CSR, impacting both the Classic and Alternative NHEJ pathways. In contrast, absence of Mre11 nuclease activities causes a milder phenotype, revealing a separation of function within the complex. We propose a model in which MRN stabilizes distant breaks and processes DNA termini to facilitate repair by both the Classical and Alternative NHEJ pathways.

The major photoproduct in UV-irradiated Bacillus spore DNA is a unique thymine dimer called spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine). The enzyme spore photoproduct lyase (SP lyase) has been found to catalyze the repair of SP dimers to thymine monomers in a reaction that requires S-adenosylmethionine. We present here the first detailed characterization of catalytically active SP lyase, which has been anaerobically purified from overexpressing Escherichia coli. Anaerobically purified SP lyase is monomeric and is red-brown in color. The purified enzyme contains ϳ3.1 iron and 3.0 acid-labile S 2؊ per protein and has a UVvisible spectrum characteristic of iron-sulfur proteins (410 nm (11.9 mM H]S-adenosylmethionine to repaired thymine is observed, providing evidence to support a mechanism in which a 5-deoxyadenosyl radical intermediate directly abstracts a hydrogen from SP C-6 to generate a substrate radical, and subsequent to radical-mediated ␤-scission, a product thymine radical abstracts a hydrogen from 5-deoxyadenosine to regenerate the 5-deoxyadenosyl radical. Together, our results support a mechanism in which S-adenosylmethionine acts as a catalytic cofactor, not a substrate, in the DNA repair reaction.The unusual resistance of bacterial spores to UV light and ionizing radiation has been of long-standing interest. In 1965, Donnellan and Setlow (1) showed that UV-irradiated bacterial spores did not contain the thymine dimers typically found in other irradiated cells but instead contained another type of thymine photoproduct. This dimer was later determined to be spore photoproduct (SP, 3 5-thyminyl-5,6-dihydrothymine; see Fig. 1), a thymine dimer that appears to be unique to Bacillus subtilis and other spore-forming microorganisms (2). The generation of these unique thymine dimers upon UV irradiation is thought to result from the binding of small, acid-soluble proteins to the spore DNA (3-7). During dormancy spores are unable to repair this damage to their DNA from UV exposure. Early in the germination cycle, however, two distinct mechanisms of DNA repair are active, presumably giving rise to the high level of UV resistance of bacterial spores. One of these mechanisms is the nucleotide excision repair pathway that detects and removes lesions from the DNA (including SP and cyclobutane-type pyrimidine dimers) in a manner similar to that characterized in Escherichia coli (8, 9). The second repair mechanism involves the specific reversal of SP to two thymines catalyzed by the enzyme SP lyase in B. subtilis (10,11).Pyrimidine dimers such as the spore photoproduct of Bacillus are a major component of UV-induced DNA damage. These dimers can block replication and transcription or can result in mutations if transcription does proceed past the region of the pyrimidine dimer. Repair of these dimers, therefore, is critical in order to avoid mutations. The only well characterized means of pyrimidine dimer repair is photoreactivation, which is catalyzed by the enzyme DNA photolyase (12, 13). The photolyase famil...

Homologous recombination (HR) facilitates accurate repair of DNA double strand breaks (DSBs) during S and G2 phases of the cell cycle by using intact sister chromatids as sequence templates. HR capacity is maximized in S and G2 by Cyclin–Dependent Kinase (CDK) phosphorylation of CtIP, which subsequently interacts with BRCA1 and the Mre11–Rad50–NBS1 (MRN) complex. Here we show that Mre11 controls these events through a direct interaction with CDK2 that is required for CtIP phosphorylation and BRCA1 interaction in normally dividing cells. CDK2 binds the C–terminus of Mre11, which is absent in an inherited allele causing Ataxia–Telangiectasia Like Disorder. This newly uncovered role for Mre11 does not require ATM activation or nuclease activities. Therefore, functions of MRN are not restricted to DNA damage responses, but include regulating HR capacity during the normal mammalian cell cycle.