在存在5-甲酰胞嘧啶(5fC)的情况下,组蛋白可以被捕获在DNA上,以形成有毒的DNA-蛋白质缀合物。它们的修复可能涉及蛋白水解消化,导致DNA-肽交联(DpCs)。这里,我们已经研究了由11聚体肽(NH2-GGGKGLGK*GGA)组成的模型DpC的复制,该肽含有与DNA中的5fC缀合的氧基赖氨酸残基(K*)。检查CXG和CXT(其中X=5fC-DpC)序列上下文。两种构建体的复制在大肠杆菌中产生低活力(<10%),而在HEK293T细胞中TLS效率高(72%)。在大肠杆菌中,DpC被绕过了很大程度上没有错误,只诱导2-3%的突变,随着SOS增加到4-5%。对于这两个序列,半靶向碱基替换是显性的,对于CXG来说,在5fC-DpC的3个碱基上,主要突变是G→T和G→C。在HEK293T细胞中,发生7-9%的突变,显性突变是CXG的半靶向G→T和CXT的T→G。这些突变在缺乏hPolη的细胞中急剧减少,hPolι或hPolζ,提示这些TLS聚合酶在诱变性病变旁路中的可能作用。使用hPolη的稳态动力学研究证实了该聚合酶在DpC的3''碱基处诱导G→T和T→G转化的能力。这项研究揭示了5fC-缀合的DpCs的独特复制模式,在大肠杆菌和人类细胞中,它们在很大程度上被绕过,并且在病变的3'位置诱导了主要的半靶向突变。
Histone proteins can become trapped on DNA in the presence of 5-formylcytosine (5fC) to form toxic DNA-protein conjugates. Their repair may involve proteolytic digestion resulting in DNA-peptide cross-links (DpCs). Here, we have investigated replication of a model DpC comprised of an 11-mer peptide (NH2-GGGKGLGK∗GGA) containing an oxy-lysine residue (K∗) conjugated to 5fC in DNA. Both CXG and CXT (where X = 5fC-DpC) sequence contexts were examined. Replication of both constructs gave low viability (<10%) in Escherichia coli, whereas TLS efficiency was high (72%) in HEK 293T cells. In E. coli, the DpC was bypassed largely error-free, inducing only 2 to 3% mutations, which increased to 4 to 5% with SOS. For both sequences, semi-targeted mutations were dominant, and for CXG, the predominant mutations were G→T and G→C at the 3\'-base to the 5fC-DpC. In HEK 293T cells, 7 to 9% mutations occurred, and the dominant mutations were the semi-targeted G → T for CXG and T → G for CXT. These mutations were reduced drastically in cells deficient in hPol η, hPol ι or hPol ζ, suggesting a role of these TLS polymerases in mutagenic TLS. Steady-state kinetics studies using hPol η confirmed that this polymerase induces G → T and T → G transversions at the base immediately 3\' to the DpC. This study reveals a unique replication pattern of 5fC-conjugated DpCs, which are bypassed largely error-free in both E. coli and human cells and induce mostly semi-targeted mutations at the 3\' position to the lesion.