DNA Damage Tolerance Mechanisms Revealed from the Analysis of Immunoglobulin V Gene Diversification in Avian DT40 Cells

Abstract

DNA replication is an essential biochemical reaction in dividing cells that frequently stalls at damaged sites. Homologous/homeologous recombination (HR)-mediated template switch and translesion DNA synthesis (TLS)-mediated bypass processes release arrested DNA replication forks. These mechanisms are pivotal for replication fork maintenance and play critical roles in DNA damage tolerance (DDT) and gap-filling. The avian DT40 B lymphocyte cell line provides an opportunity to examine HR-mediated template switch and TLS triggered by abasic sites by sequencing the constitutively diversifying immunoglobulin light-chain variable gene (IgV). During IgV diversification, activation-induced deaminase (AID) converts dC to dU, which in turn is excised by uracil DNA glycosylase and yields abasic sites within a defined window of around 500 base pairs. These abasic sites can induce gene conversion with a set of homeologous upstream pseudogenes via the HR-mediated template switch, resulting in templated mutagenesis, or can be bypassed directly by TLS, resulting in non-templated somatic hypermutation at dC/dG base pairs. In this review, we discuss recent works unveiling IgV diversification mechanisms in avian DT40 cells, which shed light on DDT mode usage in vertebrate cells and tolerance of abasic sites.

Keywords: DNA damage; abasic site; activation-induced deaminase; homologous recombination; replication; translesion DNA synthesis.

Figure 1

Schematic representation of mechanisms releasing the arrested replication fork at the damaged template. (A) Homologous recombination (HR)-mediated template switch releases the arrested replication fork using intact newly synthesized DNA as the template strand and promotes error-free bypass replication. (B) Translesion DNA synthesis (TLS) polymerases mediate direct bypass replication across the damaged template in an error-prone manner.

Figure 2

Schematic representation of the immunoglobulin variable (IgV) gene diversification mechanism in DT40 cells. The sequential actions of activation-induced deaminase (AID) and uracil DNA glycosylase (UNG) induce abasic sites in the IgV gene. The replication fork arrests at these lesions and induces template switch-mediated gene conversion with one of the 25 copies of upstream IgV pseudogenes carrying a ~10% mismatch rate, resulting in HR-mediated diversification/mutagenesis of the immunoglobulin gene (left), or TLS, resulting in somatic hypermutation at the dC/dG base pairs (right).

Figure 3

Schematic representations of the surface immunoglobulin M (sIgM) gain (A) and sIgM loss assays (B). (A) Principle of the Ig gene conversion assay. The sIgM-negative DT40 clone contains a frameshift in its rearranged V-J_λ segments, which can be repaired by pseudogene-templated conversion events. The rate of Ig gene conversion can be measured as a gain of sIgM expression in subclones by flow-cytometric analysis of sIgM staining. (B) Principle of the Ig hypermutation assay. The DT40 cells carrying wild type IgV gene, which can be disrupted by somatic hypermutation events. The rate of hypermutation events can be measured as a loss of sIgM expression in subclones by flow-cytometric analysis of sIgM staining. The sequence analysis of hypermutation event can be also carried out by selecting sIgM negative cells by cell sorter.

Figure 4

Genes involved in Ig gene conversion and hypermutation. GC and PM represent gene conversion and hypermutation, respectively. Positive, zero or negative effects by gene deletion or mutation on gene conversion and hypermutation were categorized.