Figure 4.

Synthesis from misaligned or mispaired primer–template substrates by hPolθ. (A) A portion of each primer–template substrate is shown. In each case, the primer is 20 nt long and the template is 40 nt long. The G:C substrate contains the correct bp at the primer–template junction, and extension would result in full-length product. In the C:C (−1) substrate, a C:C mispair is at the primer–template junction. The C in the primer, however, can base pair with the underlined G residue at position 21 in the template, extension from which would result in a −1 frameshift. The C:C (−2) substrate also has a C:C mispair at the primer terminus, and in this case, the C in the primer can base pair with the underlined G residue at position 22 in the template. Synthesis from this misaligned template would result in a −2 frameshift. The T:C substrate is unable to misalign; therefore, synthesis from this primer would occur by extension from the mispair and result in full-length product. (B) Extension of G:C, C:C (−1), C:C (−2), and T:C DNA substrates by E. coli Klenow fragment, hPolθ, or hPolη. Lanes 1–4, extension of each substrate by Klenow fragment; lanes 5–8, extension of each substrate by hPolθ; and lanes 9–12, extension of each substrate by hPolη. Lengths of primer extension products are indicated on the left.