D39V.2bitpart of Mazzuoli, van Raaphorst et. al, 2024

Nucleoid-associated proteins (NAPs) are crucial for maintaining chromosomal compaction and architecture and are actively involved in DNA replication, recombination, repair, and gene regulation. In the opportunistic pathogen <em>Streptococcus pneumoniae,</em> HU is the only identified NAP, and its role in chromosome conformation and other essential processes has not yet been investigated. Here, we use a multi-scale approach to explore the role of HU in chromosome conformation and segregation dynamics. By combining superresolution microscopy and whole-genome binding analysis, we describe the nucleoid as a dynamic structure where HU binds transiently across the entire nucleoid, with a preference for the origin of replication over the terminus. Reducing cellular HU levels impacts nucleoid maintenance and disrupts robust nucleoid scaling with cell size. This effect is similar to the distortion caused by fluoroquinolone-antibiotics, supporting earlier observations that HU is essential for maintaining DNA supercoiling. Furthermore, in cells lacking HU, the replication machinery is misplaced, and cells are unable to initiate and proceed with on-going replication. Chromosome conformation capture (Hi-C) experiments revealed that HU is required to maintain cohesion between the two chromosomal arms, in a similar way to the structural maintenance of the chromosome complex SMC. Together, we show that by promoting long-range chromosome interactions and supporting the architecture of the domain encompassing the origin, HU is fundamental for chromosome integrity and the intimately related processes of chromosome replication and segregation.

类核关联蛋白（nucleoid-associated proteins, NAPs）是维持染色体压缩状态与三维结构的核心因子，且主动参与DNA复制、重组、修复及基因调控等关键生物学过程。在机会致病菌肺炎链球菌（*Streptococcus pneumoniae*）中，HU是目前已鉴定的唯一类核关联蛋白，其在染色体构象维持及其他核心生理过程中的作用尚未被研究。本研究采用多尺度研究策略，探究HU在染色体构象与分离动态中的功能。通过结合超分辨率显微成像技术与全基因组结合分析，我们发现类核是一种动态结构：HU可在整个类核区域瞬时结合，且相较于复制终点，对复制起点具有显著的结合偏好性。降低细胞内HU的表达水平会破坏类核的稳定维持，并扰乱类核与细胞大小间的正常缩放关系。该效应与氟喹诺酮类抗生素引发的染色体构象畸变相似，佐证了此前关于HU对维持DNA超螺旋至关重要的观测结论。此外，在HU缺失的细胞中，DNA复制机器的定位出现异常，细胞无法启动并维持正常的DNA复制进程。染色体构象捕获（Hi-C）实验结果显示，HU对于维持两条染色体臂之间的黏连是必需的，其功能与染色体结构维持复合体（structural maintenance of chromosomes, SMC）类似。综上，本研究证实HU通过促进长程染色体相互作用、维持包含复制起点的染色体结构域的架构，对染色体完整性以及与之密切相关的染色体复制与分离过程发挥关键作用。