Roles of Type 1A Topoisomerases in Genome Maintenance in Escherichia coli

In eukaryotes, type 1A topoisomerases (topos) act with RecQ-like helicases to maintain the stability of the genome. Despite having been the first type 1A enzymes to be discovered, much less is known about the involvement of the E. coli topo I (topA) and III (topB) enzymes in genome maintenance. These enzymes are thought to have distinct cellular functions: topo I regulates supercoiling and R-loop formation, and topo III is involved in chromosome segregation. To better characterize their roles in genome maintenance, we have used genetic approaches including suppressor screens, combined with microscopy for the examination of cell morphology and nucleoid shape. We show that topA mutants can suffer from growth-inhibitory and supercoiling-dependent chromosome segregation defects. These problems are corrected by deleting recA or recQ but not by deleting recJ or recO, indicating that the RecF pathway is not involved. Rather, our data suggest that RecQ acts with a type 1A topo on RecA-generated recombination intermediates because: 1-topo III overproduction corrects the defects and 2-recQ deletion and topo IIII overproduction are epistatic to recA deletion. The segregation defects are also linked to over-replication, as they are significantly alleviated by an oriC::aph suppressor mutation which is oriC-competent in topA null but not in isogenic topA+ cells. When both topo I and topo III are missing, excess supercoiling triggers growth inhibition that correlates with the formation of extremely long filaments fully packed with unsegregated and diffuse DNA. These phenotypes are likely related to replication from R-loops as they are corrected by overproducing RNase HI or by genetic suppressors of double topA rnhA mutants affecting constitutive stable DNA replication, dnaT::aph and rne::aph, which initiates from R-loops. Thus, bacterial type 1A topos maintain the stability of the genome (i) by preventing over-replication originating from oriC (topo I alone) and R-loops and (ii) by acting with RecQ.

在真核生物中，1A型拓扑异构酶（type 1A topoisomerases，以下简称topos）与RecQ样解旋酶协同作用，维持基因组稳定性。尽管它们是首批被发现的1A型酶，但学界对大肠杆菌（E. coli）拓扑异构酶I（topo I，由topA基因编码）与拓扑异构酶III（topo III，由topB基因编码）在基因组维持中的作用仍知之甚少。现有研究认为这两种酶具备截然不同的细胞功能：拓扑异构酶I可调控DNA超螺旋与R环形成，而拓扑异构酶III则参与染色体分离过程。为更深入地表征二者在基因组维持中的功能，本研究采用了包括抑制子筛选在内的遗传学手段，并结合显微镜技术观察细胞形态与类核结构。研究发现，topA基因突变株会出现生长抑制与依赖DNA超螺旋的染色体分离缺陷。敲除recA或recQ可修复此类缺陷，但敲除recJ或recO则无修复效果，提示RecF通路未参与该过程。相反，本研究数据表明RecQ与1A型拓扑异构酶协同作用于RecA介导的重组中间产物，依据如下：其一，过量表达拓扑异构酶III可修复上述缺陷；其二，recQ敲除与拓扑异构酶III过量表达对recA敲除存在上位性效应。染色体分离缺陷还与复制过度相关：oriC::aph抑制子突变可显著缓解该缺陷——该突变在topA基因敲除株中仍保有oriC功能，但在同基因背景的topA野生型细胞中则无此活性。当拓扑异构酶I与拓扑异构酶III同时缺失时，过量DNA超螺旋会引发生长抑制，该现象与极度伸长的丝状细胞形成密切相关，此类丝状细胞内充满未分离且弥散的DNA。这类表型大概率与R环介导的复制过程相关，因为过量表达RNase HI，或是靶向topA与rnhA双突变株中影响组成型稳定DNA复制（该复制起始于R环）的遗传抑制子dnaT::aph与rne::aph，均可修复上述表型。综上，细菌1A型拓扑异构酶通过两种途径维持基因组稳定性：其一，阻止源自oriC（仅由拓扑异构酶I介导）与R环的过度复制；其二，与RecQ协同发挥作用。