Mechanistic basis for microhomology identification and genome scarring by polymerase theta

Abstract

DNA polymerase theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3′ single-stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3′ terminus and favoring bidirectional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3 to 30 bp of sequence that is identical to flanking DNA (“templated” insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification and shows how it both mitigates limitations implicit in the microhomology requirement and generates distinctive genomic scars associated with pathogenic genome instability.

Regulation of Error-Prone DNA Double-Strand Break Repair and Its Impact on Genome Evolution

“…In one study using mammalian cells, the efficiency of TMEJ was high when 2-4 bp microhomologies were directly adjacent to a DSB but dropped significantly when the distance from the break was increased to 10-30 bp. Increasing the length of microhomology to 6 bp rescued this distance-dependent decrease, illustrating that successful alt-EJ depends on a balance between the size of the microhomology and its location relative to the break [37,86].…”

“…Evidence from studies using mouse embryonic fibroblasts with Cas9-generated DSBs suggests pol theta can sample all microhomologous sequences for annealing within the 3 terminal 25 nucleotides of a pair of ends [10]. Iterative rounds of the annealing, synthesis, and dissociation steps can lead to insertions templated from regions around the DSB [20,32,37].…”

“…This study also found that doubling an AT-rich microhomology from 3 bp to 6 bp increased the efficiency of MMEJ, highlighting a balance between microhomology length and AT/GC content. For alt-EJ outcomes that involved insertions, flanking sequences with 80% AT content produced more insertions than sequences with 80% GC content [37]. This bias may be due to the propensity of AT-rich microhomologies to dissociate following the initial MMEJ repair attempt, leading to SD-MMEJ and insertion formation.…”

“…These events were likely due to the fill-in of Cas9-induced DSBs with single nucleotide 5' overhangs. In another study of Cas9-induced breaks, 5-10% were repaired by TMEJ under normal physiological conditions [37]. Thus, it appears clear that the ability to predict alt-EJ repair products will be critical to reach the goal of understanding the potential outcomes of Cas9 genome editing experiments.…”

BRCA1-associated structural variations are a consequence of polymerase theta-mediated end-joining

“…A dependence on polymerase theta for HR-deficient cells was noticed earlier in mouse embryonic fibroblasts 51 tumour cells: HR-deficient epithelial ovarian cancer cells depend on TMEJ 52 , whereas similar effects were recently observed for cisplatin-resistant lung cancer cells (which had upregulated polymerase theta expression) 53 . Moreover, it was recently shown that tumours with BRCA mutations show a significantly higher frequency of templated insertions, which is a hallmark of polymerase theta action, than BRCA proficient tumours 54 . The dependency of HR-deficient cancer cells on TMEJ point to polymerase theta as an interesting therapeutic strategy for a specific set of tumours.…”