Replication of [AT/TA] microsatellite sequences by human DNA polymerase δ holoenzymes is dependent on dNTP and RPA levels.

Difficult-to-Replicate Sequences (DiToRS) are natural impediments in the human genome that inhibit DNA replication under endogenous replication. Some of the most widely-studied DiToRS are A+T-rich, high "flexibility regions," including long stretches of perfect [AT/TA] microsatellite repeats that have the potential to collapse into hairpin structures when in single-stranded DNA (ssDNA) form and are sites of recurrent structural variation and double-stranded DNA (dsDNA) breaks. Currently, it is unclear how these flexibility regions impact DNA replication, greatly limiting our fundamental understanding of human genome stability. To investigate replication through flexibility regions, we utilized FRET to characterize the effects of the major ssDNA-binding complex, RPA, on the structure of perfect [AT/TA] microsatellite repeats and also re-constituted human lagging strand replication to quantitatively characterize initial encounters of pol δ holoenzymes with A+T-rich DNA template sequences. The results indicate that [AT/TA] sequences adopt hairpin structures that are unwound by RPA and pol δ holoenzymes support dNTP incorporation through the [AT/TA] sequences as well as an A+T-rich, non-structure forming sequence. Furthermore, the extent of dNTP incorporation is dependent on the sequence of the DNA template and the concentration of dNTPs. Importantly, the effects of RPA on the replication of [AT/TA] sequences are dependent on the concentration of dNTPs, whereas the effects of RPA on the replication of an A+T-rich, non-structure forming sequence are independent of dNTP concentration. Collectively, these results reveal complexities in lagging strand replication and provide novel insights into how flexibility regions contribute to genome instability.