Lesion bypass then occurs by FANCD2/I-dependent nucleotide insertion across the damaged template base followed by polymerase ζ-dependent extension.
These lesions include interstrand crosslinks, ssDNA gaps left behind the replication fork, and double-strand DNA breaks (DSB) due to DNA damaging agents that cause replication to occur through damaged template or by programmed events (e.g., during meiosis) [1,2].
To overcome the replicative impasse that is posed by DNA lesions, cells can employ two general strategies: either halt cell cycle progression to allow time for repair or read-through and bypass the damaged template.
Incorporation of dATP also predominates during the bypass of 1, N-εdG by E. coli polymerases pol I exo– and pol II exo– using a 5′-T XT-3′ damaged template, also containing the template 5′-neighbor T. (42) Other polymerases, however, may use different mechanisms to bypass the lesion.
Given a diploid genome size of 6 pg, this suggests a single molecule PCR efficiency of ∼50% for filled sites and ∼100% for empty sites, and demonstrates that the low yield of filled-site PCR products (Fig. 3) is mainly due to inefficient amplification of long DNA molecules, rather than damaged template molecules.
Therefore, it is likely that the DNA replication machinery frequently encounters damaged templates giving rise to stalled replication forks.
Furthermore, these variants negatively affected error-free replication across damaged templates, as observed by a reduction in the X structures representing damage-bypass SCJs.
Replicative DNA polymerases are intrinsically more accurate but also cannot process templates with lesions or damage, while lesion-repair polymerases can process damaged templates but do not have good intrinsic fidelity.
A number of studies also associate Smc5/6 with repair of stalled/collapsed replication forks since replication through damaged templates caused an accumulation of X-shaped DNA structures in smc5/6 mutants [ 5, 6, 7].
Methodologies that assess, minimise or remove formalin-induced DNA damaged templates as part of MPS protocols will aid in the interpretation of genomic results leading to better patient outcomes.
