These results suggest thatVgenes have inherent properties that affect their rate of repair, which might allow misinserted nucleotides to linger longer. The canonical base excision repair pathway could remove U produced by AID. during repair synthesis, but the other proteins are not necessary. Second, we analysed the role of low-fidelity DNA polymerases , and in synthesizing mutations, and conclude that polymerase is the dominant participant by generating mutations at A:T base pairs. In the non-mutagenic pathway, we examined the role of the Cockayne syndrome B protein that interacts with other repair proteins. Mice deficient in this protein had normal hypermutation and class switch recombination, showing that it is not involved. Keywords:immunoglobulin genes, somatic hypermutation, class switch recombination, MSH2MSH6, DNA polymerase , Cockayne syndrome B == 1. Aberrant DNA repair generates antibody diversity == The identification of the activation-induced cytidine deaminase (AID) protein by Honjo and colleagues (Muramatsuet al. 2000), and the discovery that it deaminates cytosine to uracil in immunoglobulin genes (Di Noia & Neuberger 2002), has exploded our knowledge of how antibodies become diversified. AID Nepicastat (free base) (SYN-117) initiates both affinity maturation in variable (V) genes and class switch recombination (CSR) of heavy chain constant (CH) genes. Deamination of cytosine is mutagenic, and it can be processed by a mutagenic pathway to generate mutations, or by a non-mutagenic pathway to remove mutations (figure 1). Both these pathways probably occur at the same time in activated B cells, so that the ultimate outcome is a balance between the mutation and the repair. == Figure 1. == Mutagenic and non-mutagenic pathways for handling U. The mutagenic pathway involves either removal of U by uracil DNA glycosylase (UNG), or recognition of U:G by MSH2MSH6. Mutations arise by DNA polymerases copying the abasic site or polymerase synthesizing mutations opposite A and T nucleotides in a gap generated by exonuclease 1. Strand Nepicastat (free base) (SYN-117) breaks occur when the abasic site is nicked by an APE, or by an endonuclease associated with MMR. The non-mutagenic pathway removes U with UNG and APE1, and polymerase synthesizes the correct Nepicastat (free base) (SYN-117) nucleotide (CSB, Cockayne syndrome B). In the mutagenic pathway, uracil (U) can be Nepicastat (free base) (SYN-117) processed by several enzymes that have been identified based on genetic and biochemical evidence (Di Noia & Neuberger 2002;Petersen-Mahrtet al. 2002;Radaet al. 2002). First, U could be replicated by high-fidelity DNA Rabbit Polyclonal to C-RAF (phospho-Ser301) polymerases to produce C:G transitions. Second, U could be removed by uracil DNA glycosylase (UNG) to produce an abasic site, which when copied by low-fidelity DNA polymerases, would generate C:G transitions and transversions. Third, U could be recognized as a U:G mismatch by the MSH2MSH6 mismatch repair (MMR) proteins, which would generate a gap that could be filled in by the low-fidelity DNA polymerase (pol) to produce mutations of A:T base pairs (bp;Zenget al. 2001;Wilsonet al. 2005). The last two steps are also associated with strand breaks, which produce substrates for recombination during heavy-chain class switching. In the non-mutagenic pathway, which consists of base excision repair proteins, U is removed by UNG to produce an abasic site. The abasic site is cleaved by Nepicastat (free base) (SYN-117) the apurinic/apyrimidinic endonuclease (APE) 1, and the deoxyribose phosphate group is removed by DNA pol , which then fills in the one nucleotide gap with high fidelity. This pathway is present in every cell, and is used to remove uracils generated by spontaneous deamination. No mutations or strand breaks would be produced, and heavy-chain class switching would be diminished. Thus, in B cells, AID generates many uracils in a defined region around the rearrangedVgene and in the switch regions preceding eachCHgene. Presumably not all the uracils are repaired, and those that are left in the DNA would produce nucleotide substitutions by the mutagenic pathway described above. We have studied proteins that are involved in the mutagenic pathway by examining mice that are deficient in the proteins, and by analysing the biochemical interactions between the relevant proteins. Intriguingly, the hypermutation machinery has hijacked certain repair proteins from the MMR and base excision pathways to create mutations, rather than repair them. == 2. mismatch repair proteins == In the mutagenic pathway, many mismatches could be generated before they are fixed as mutations after DNA replication. In eukaryotic MMR, the heterodimer MSH2MSH6 binds to single-nucleotide mismatches, and MSH2MSH3 binds to loops created by insertions, deletions or mispairing on one strand. The heterodimer PMS2MLH1 is then recruited to the complex. PMS2 has recently been shown to function as an endonuclease (Kadyrovet al. 2006), which nicks the.