Ribonucleotides are frequently incorporated into DNA and can be used as a marker of DNA replication enzymology. To investigate on a genome-wide scale, how E. coli pol V accesses undamaged chromosomal DNA during the SOS response, we mapped the location of ribonucleotides incorporated by steric gate variants of pol V across the entire E. coli genome. To do so, we used strains that are deficient in ribonucleotide excision repair (ΔrnhB), deficient in pol IV DNA polymerase, constitutively express all SOS-regulated genes [lexA(Def)] and constitutively “activated” RecA* (recA730). The strains also harbor two steric gate variants of E. coli pol V (Y11A, or F10L), or a homolog of pol V, (pol V_R391-Y13A). Ribonucleotides are frequently incorporated by the pol V-Y11A and pol V_R391-Y13A variants, with a preference to the lagging strand. In contrast, the pol V-F10L variant incorporates less ribonucleotides and no strand preference is observed. Sharp transitions in strand specificity are observed at the replication origin (oriC), while a gradient is observed at the termination region. To activate RecA* in a recA⁺ strain, we treated the strains with ciprofloxacin and genome-wide mapped the location of the incorporated ribonucleotides. Again, the pol V-Y11A steric gate variant exhibited a lagging strand preference. Our data are consistent with a specific role for pol V in lagging strand DNA synthesis across the entire E. coli genome during the SOS response.

核糖核苷酸经常掺入 DNA 中，可用作 DNA 复制酶学的标记。为了在全基因组范围内研究大肠杆菌pol V如何在 SOS 响应期间访问未受损的染色体 DNA，我们绘制了整个大肠杆菌基因组中由 pol V 的空间门变体掺入的核糖核苷酸的位置。为此，我们使用了核糖核苷酸切除修复缺陷 (Δ rnhB )、pol IV DNA 聚合酶缺陷、组成型表达所有 SOS 调节基因 [ lexA (Def)] 和组成型“激活”的 RecA* ( recA730 ) 的菌株。这些菌株还含有大肠杆菌的两种空间门变体pol V（Y11A，或 F10L），或 pol V 的同系物，（pol V _R391 -Y13A）。核糖核苷酸经常被 pol V-Y11A 和 pol V _R391 -Y13A 变体掺入，优先于滞后链。相比之下，pol V-F10L 变体包含较少的核糖核苷酸，并且没有观察到链偏好。在复制起点 ( oriC )处观察到链特异性的急剧转变，而在终止区域处观察到梯度。在recA ^{+ 中}激活 RecA*菌株，我们用环丙沙星处理菌株，并在全基因组范围内绘制了掺入核糖核苷酸的位置。同样，pol V-Y11A 立体门变体表现出滞后链偏好。我们的数据与 pol V 在SOS 响应期间整个大肠杆菌基因组的滞后链 DNA 合成中的特定作用一致。