The very 5′ end and the constant region of Ig genes are spared from somatic mutation because AID does not access these regions

Somatic hypermutation (SHM) is restricted to VDJ regions and their adjacent flanks in immunoglobulin (Ig) genes, whereas constant regions are spared. Mutations occur after about 100 nucleotides downstream of the promoter and extend to 1–2 kb. We have asked why the very 5′ and most of the 3′ region of Ig genes are unmutated. Does the activation-induced cytosine deaminase (AID) that initiates SHM not gain access to these regions, or does AID gain access, but the resulting uracils are repaired error-free because error-prone repair does not gain access? The distribution of mutations was compared between uracil DNA glycosylase (Ung)-deficient and wild-type mice in endogenous Ig genes and in an Ig transgene. If AID gains access to the 5′ and 3′ regions that are unmutated in wild-type mice, one would expect an “AID footprint,” namely transition mutations from C and G in Ung-deficient mice in the regions normally devoid of SHM. We find that the distribution of total mutations and transitions from C and G is indistinguishable in wild-type and Ung-deficient mice. Thus, AID does not gain access to the 5′ and constant regions of Ig genes. The implications for the role of transcription and Ung in SHM are discussed

The Fanconi anemia proteins FANCD2 and FANCJ interact and regulate each other's chromatin localization.

Fanconi anemia is a genetic disease resulting in bone marrow failure, birth defects, and cancer that is thought to encompass a defect in maintenance of genomic stability. Mutations in 16 genes (FANCA, B, C, D1, D2, E, F, G, I, J, L, M, N, O, P, and Q) have been identified in patients, with the Fanconi anemia subtype J (FA-J) resulting from homozygous mutations in the FANCJ gene. Here, we describe the direct interaction of FANCD2 with FANCJ. We demonstrate the interaction of FANCD2 and FANCJ in vivo and in vitro by immunoprecipitation in crude cell lysates and from fractions after gel filtration and with baculovirally expressed proteins. Mutation of the monoubiquitination site of FANCD2 (K561R) preserves interaction with FANCJ constitutively in a manner that impedes proper chromatin localization of FANCJ. FANCJ is necessary for FANCD2 chromatin loading and focus formation in response to mitomycin C treatment. Our results suggest not only that FANCD2 regulates FANCJ chromatin localization but also that FANCJ is necessary for efficient loading of FANCD2 onto chromatin following DNA damage caused by mitomycin C treatment

Promotion of RAD51-Mediated Homologous DNA Pairing by the RAD51AP1-UAF1 Complex

The UAF1-USP1 complex deubiquitinates FANCD2 during execution of the Fanconi anemia DNA damage response pathway. As such, UAF1 depletion results in persistent FANCD2 ubiquitination and DNA damage hypersensitivity. UAF1-deficient cells are also impaired for DNA repair by homologous recombination. Herein, we show that UAF1 binds DNA and forms a dimeric complex with RAD51AP1, an accessory factor of the RAD51 recombinase, and a trimeric complex with RAD51 through RAD51AP1. Two small ubiquitin-like modifier (SUMO)-like domains in UAF1 and a SUMO-interacting motif in RAD51AP1 mediate complex formation. Importantly, UAF1 enhances RAD51-mediated homologous DNA pairing in a manner that is dependent on complex formation with RAD51AP1 but independent of USP1. Mechanistically, RAD51AP1-UAF1 co-operates with RAD51 to assemble the synaptic complex, a critical nucleoprotein intermediate in homologous recombination, and cellular studies reveal the biological significance of the RAD51AP1-UAF1 protein complex. Our findings provide insights into an apparently USP1-independent role of UAF1 in genome maintenance