Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition. Shastri, Tsai et al.

Published: 10-10-2018| Version 1 | DOI: 10.17632/9gwyx4vxt8.1
Contributor:
Nishita Shastri

Description

In this manuscript, we identify new classes of difficult-to-replicate sequences in the mouse and human genomes that are highly dependent on ATR function for stability during DNA replication. Structure-forming short tandem repeats, inverted retroelements, and quasi- palindromic AT-rich repeats characterize the sites for fork collapse caused by ATR inhibition. The data deposited here include original images for the 2D gels, PAGE gels, and Southern blots presented in the manuscript. These include non-denaturing PAGE gels of oligonucleotide repeats most commonly observed within RPA-enriched sites from ATR inhibition that demonstrate single-band formation and greater electrophoretic mobility than expected based on their length, suggesting formation of compact, uniquely folded structures (Figure 3A and G). Also included are 2D neutral-neutral gel electrophoresis images of synthesis occurring across inserted (CAGAGG)105 repeats or a scrambled sequence in an orientation that is either proximal to the bidirectional SV40 origin (ori-proximal), or distal (ori-distal) in a pML113 vector transfected into U2OS cells. Southern hybridization to the probe indicated in Figure 4D and E was performed. These gels show double-Y migration products only in (CAGAGG)105 repeat-containing vectors, not scrambled controls, indicating that fork stalling occurs at the (CAGAGG)105 repeat interface from both sides. This inhibition of fork movement was increased 5-fold upon aphidicolin treatment. These images refer to data presented in Figure 4 and Figure S5. To prove sufficiency of the (CAGAGG)n repeat in causing breaks, an HFUGW vector containing (CAGAGG)105 repeats or a scrambled sequence was transfected into 4-3 MEFs and assayed for breakage at insert-proximal vector sequences by BrITL qRT-PCR. The TAE agarose gel depicts PstI-digested HFUGW vector prior to transfection into cells, with insert sequences from (CAGAGG)105 or scrambled control indicated above each lane. A Southern blot depicts the (CAGAGG)105 and scrambled insert sequence detected after transfection of cells with the relevant insert-containing vector. These images refer to data presented in Figure 6 and Figure S6.

Files