Figure 1
Effects of UV induced replication stalling in HEK293T cells. (A) A schematic map of human mitochondrial DNA (mtDNA) showing the DraI restriction sites adjacent to the non-coding region (NCR); other DraI sites are omitted for clarity. Different forms of the DraI fragment downstream of the origin of H-strand replication (OH) were analyzed using two-dimensional agarose gel electrophoresis (2D-AGE). Location of the probe used for Southern hybridization as indicated. (B) Illustration of the different mtDNA forms detected by the probe. Linear forms spanning one restriction fragment length (1n; nts 12,273–16,012) are the most abundant. As OH is not included in the analyzed region, asynchronous (or so-called strand-displacement) replication forks initiating from this origin enter the fragment as they proceed, resulting in slow-moving y-forms (smy’s) due to the inability of the restriction enzyme to cut the displaced strand. If replication intermediates are fully double-stranded DNA, both leading and lagging strand are cut, resulting in regular y-forms. Replication bubbles originating from replication initiation within the DraI fragment will migrate on the bubble arc (b). Recombination junctions consist of two restriction fragment-sized linear molecules and form an x-spike that starts from the 2n position of the linear arc. Regressed replication forks (rx) form specific chicken foot structures growing from the y-arc30. (C) 2D-AGE of the DraI 12,273–16,012 fragment of mtDNA from HEK293T cells before (untreated) and 4 h after 30 s exposure to 1.34 mJ/cm2, 302 nm wavelength UVB (UV treated). S1 nuclease treatment (panels with gray background) was applied to reveal fully double-stranded DNA (dsDNA) forms. Note the appearance of the different x-forms, replication bubble (b) and the overall increase in dsDNA intermediates. (D) MGME1 overexpression (OE) reduces all x-forms, whereas knockout (KO) of MGME1 has an opposite effect along with disappearance of the bubble arc.
