Activities of human exonuclease 1 that promote cleavage of transcribed immunoglobulin switch regions

Abstract

Eukaryotic exonuclease 1 functions in replication, recombination, mismatch repair, telomere maintenance, immunoglobulin (Ig) gene class switch recombination, and somatic hypermutation. The enzyme has 5-3 exonuclease, flap endonuclease, and weak RNaseH activity in vitro, but it has been difficult to reconcile these activities with its diverse biological functions. We report robust cleavage by human exonuclease 1 of transcribed G-rich DNA sequences with potential to form G loops and G4 DNA. Predicted Ig switch recombination intermediates are substrates for both exonucleolytic and 5 flap endonucleolytic cleavage. Excision is nick-dependent and structure-dependent. These results lead to a model for exonuclease 1 function in class switch recombination in which cleavage at activation-induced deaminase (AID)-initiated nicks produces gaps that become substrates for further attack by AID and subsequent repair.antibody ͉ B cell ͉ cotranscriptional hybrid ͉ G4 DNA ͉ switch recombination E ukaryotic exonuclease 1 (EXO1) belongs to class III of the RAD2 family of nucleases, which includes structure-specific nucleases like flap endonuclease 1 (FEN1). Purified recombinant human EXO1 (hEXO1) possesses 5Ј-3Ј exonuclease activity on dsDNA, 5Ј flap endonuclease activity, and weak RNaseH activity, and it is essential for both 5Ј and 3Ј nick-directed mismatch repair (1-4). In Saccharomyces cerevisiae, EXO1 functions in mismatch repair, replication, recombination, and at the telomeres (5-8). Mammalian EXO1 was identified as a homolog of S. cerevisiae Exo1p (1, 9) and by virtue of its interaction with hMSH2 (10). hEXO1 functionally complements its yeast homolog, and hEXO1 overexpression rescues conditional lethality of S. cerevisiae rad27 (FEN1) mutants (2), suggesting some overlap of functions of EXO1 and FEN1. Exo1 Ϫ/Ϫ mice display impaired meiosis, microsatellite instability, increased level of mutations and frequency of lymphomas (11), diminished efficiency of immunoglobulin (Ig) class switch recombination, and an altered spectrum of somatic hypermutation (12). In mice with dysfunctional telomeres, EXO1-deficiency extends life span and reduces genomic instability (13).Class switch recombination is a regulated process of DNA deletion that joins a new constant region to the rearranged and expressed Ig heavy chain variable region in activated B cells (14-16). Recombination junctions are within switch (S) regions, 2-to 10-kb noncoding regions that are transcribed from a dedicated upstream promoter to activate recombination (Fig. 1A). Recombination is initiated by C to U deamination by the B cell-specific enzyme, activation-induced deaminase (AID), which preferentially deaminates within the consensus motif, WRCY (W ϭ A/T, R ϭ purine, Y ϭ pyrimidine). AID-initiated damage is processed by redundant pathways, dependent on either uracil excision by UNG or mismatch recognition by MutS␣ (17). DNA nicking may then be promoted by the MRE11 AP lyase (18) or other AP endonucleases and lyases (19) or by factors that nick DNA for mismatch repair ...

Distinct Activities of Exonuclease 1 and Flap Endonuclease 1 at Telomeric G4 DNA

“…6B, upper; Fig. 6C), comparable to hEXO1 activity at transcribed Ig switch regions [42]. hEXO1 was also somewhat active on the nicked substrate, consistent with its documented activity on nicked DNA in mismatch repair.…”

Combinatorial H3K9acS10ph Histone Modification in IgH Locus S Regions Targets 14-3-3 Adaptors and AID to Specify Antibody Class-Switch DNA Recombination

“…Nearby SSBs on opposite strands of S region “core” can readily form staggered DSBs, obligatory intermediates of CSR. Conversion of SSBs to DSBs in the flanking area of S region depends on mismatch repair proteins (MMRs), such as MSH2 and MSH6 and exonuclease 1 (EXO1) (Vallur and Maizels, 2008). In addition to providing substrates for AID DNA deamination, S region 5’-AGCT-3’ repeats are bound with high affinity by 14-3-3 adaptors, which function as scaffolds to stabilize AID on S region DNA (Xu et al, 2010) – the REV1 DNA polymerase functions as a scaffold protein to recruit/stabilize UNG (Zan et al, 2012).…”

Putting together the pieces: evolutionary mechanisms at work within genomes

“…The immunoglobulin class switch demonstrates that environmental signals can trigger focused genome reorganization. (13) As we continue to analyze full genome sequences, we will come to be in a position to understand the ''wiring'' enough to consider whether and how distinct types of stress might influence genome variation in the metazoan germ line. As has been discussed in the context of bacterial evolution (14) it is necessary to clarify whether an observed increase in variation is induced by stress, is an inevitable consequence of increased genetic damage and decreased repair caused by stress, or whether variation is selected from a subpopulation that is more mutable and thus able to adapt more quickly during stress.…”