Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO 63110, USA.
Nucleic Acids Res. 2019 Sep 19;47(16):8595-8605. doi: 10.1093/nar/gkz608.
G-quadruplexes (G4s) are stable secondary structures that can lead to the stalling of replication forks and cause genomic instability. Pif1 is a 5' to 3' helicase, localized to both the mitochondria and nucleus that can unwind G4s in vitro and prevent fork stalling at G4 forming sequences in vivo. Using in vitro primer extension assays, we show that both G4s and stable hairpins form barriers to nuclear and mitochondrial DNA polymerases δ and γ, respectively. However, while single-stranded DNA binding proteins (SSBs) readily promote replication through hairpins, SSBs are only effective in promoting replication through weak G4s. Using a series of G4s with increasing stabilities, we reveal a threshold above which G4 through-replication is inhibited even with SSBs present, and Pif1 helicase is required. Because Pif1 moves along the template strand with a 5'-3'-directionality, head-on collisions between Pif1 and polymerase δ or γ result in the stimulation of their 3'-exonuclease activity. Both nuclear RPA and mitochondrial SSB play a protective role during DNA replication by preventing excessive DNA degradation caused by the helicase-polymerase conflict.
四链体（G4s）是稳定的二级结构，可导致复制叉停滞，并引起基因组不稳定。Pif1 是一种 5' 到 3' 的解旋酶，定位于线粒体和细胞核中，能够在体外解开 G4s，并防止体内 G4 形成序列的叉停滞。通过体外引物延伸测定，我们发现 G4s 和稳定的发夹结构分别构成核和线粒体 DNA 聚合酶 δ 和 γ 的障碍。然而，尽管单链结合蛋白 (SSBs) 容易促进发夹结构的复制，但 SSBs 仅在促进弱 G4s 的复制时才有效。通过一系列稳定性增加的 G4s，我们揭示了一个阈值，超过该阈值，即使存在 SSBs，G4 也会被抑制，并且需要 Pif1 解旋酶。由于 Pif1 沿着模板链以 5'-3' 的方向移动，Pif1 和聚合酶 δ 或 γ 之间的正面碰撞会刺激它们的 3'-外切核酸酶活性。核 RPA 和线粒体 SSB 通过防止解旋酶-聚合酶冲突引起的过度 DNA 降解，在 DNA 复制过程中发挥保护作用。
