Unchanged PCNA and DNMT1 dynamics during replication in DNA ligase I-deficient cells but abnormal chromatin levels of non-replicative histone H1

**Background** DNA ligase I (LigI) is essential for joining Okazaki fragments during lagging-strand DNA replication. In *S. cerevisiae*, ligation by Cdc9 is coupled with PCNA unloading and nucleosome assembly. Mammalian cells have a backup ligase, LigIIIα/XRCC1, which can join Okazaki fragments in the absence of LigI. LigI also interacts with UHRF1 and DNMT1 to promote maintenance DNA methylation. The study aimed to investigate the dynamics of replication proteins, DNA methylation machinery, and histone deposition in LigI-deficient mammalian cells.

**Methods** The study used human *lig1* mutant fibroblasts (46BR.1G1) and mouse *LIG1* null B-cell lymphoma cells (CH12F3). Protein dynamics on newly synthesized DNA were analyzed using accelerated native isolation of proteins on nascent DNA (aniPOND) combined with quantitative immunoblotting and stable isotope labeling by amino acids in cell culture (SILAC) followed by mass spectrometry. Cells were pulse-labeled with EdU for 15 minutes, followed by thymidine chases of 0, 30, and 60 minutes. Proteins associated with EdU-labeled DNA were affinity purified and quantified. Bulk chromatin levels were assessed by subcellular fractionation and immunoblotting.

**Key Results** 1. **PCNA dynamics unchanged**: In both human and mouse LigI-deficient cells, PCNA association with and dissociation from newly synthesized DNA was not altered compared to wild-type cells (Fig. 1b,d). SILAC-MS confirmed no differences in PCNA, RFC1, ATAD5, or RFC2-5 kinetics (Fig. 2). 2. **Increased Pol δ association**: There was increased association of DNA polymerase δ (Pol δ) with newly synthesized DNA and bulk chromatin in *LIG1* null cells, while Pol ε levels were unchanged (Fig. 3). 3. **LigIIIα/XRCC1 recruitment**: XRCC1 showed increased association with newly synthesized DNA and bulk chromatin in *LIG1* null cells (Fig. 4a,c). LigIIIα levels were elevated in bulk chromatin (Fig. 4c,e), despite similar total protein levels (Fig. S3). 4. **DNMT1 and UHRF1 dynamics intact**: The recruitment and dissociation of DNMT1, UHRF1, G9a, and GLP from newly replicated DNA were not altered in *LIG1* null cells (Fig. 5a-d). Chromatin levels of these proteins were similar between wild-type and null cells (Fig. 5e, Fig. S2). 5. **Histone H1.0 enrichment**: The replication-independent histone H1 variant H1.0 was significantly increased on newly synthesized DNA and bulk chromatin in *LIG1* null cells (Fig. 6a,b). Replication-dependent H1 variants (H1.1, H1.2, H1.4, H1.5) were slightly decreased (Fig. 6b). Immunoblotting confirmed elevated H1.0 in chromatin of mouse *LIG1* null cells and human 46BR.1G1 cells (Fig. 7a,b). Expression of wild-type LigI did not reduce H1.0 levels, indicating a stable epigenetic change (Fig. 7a,b, Fig. S5).

**Clinical Implications** The study demonstrates that LigI deficiency does not disrupt PCNA or DNMT1 dynamics at replication forks, likely due to effective backup by LigIIIα/XRCC1. This supports the potential use of LigI inhibitors as cytostatic agents with limited toxicity to normal tissues. The stable increase in histone H1.0 chromatin levels in LigI-deficient cells represents an epigenetic alteration that may contribute to immune dysfunction in inherited LigI deficiency syndromes and could influence cancer cell behavior, such as chemotherapy resistance. The findings highlight the need for further studies on acute LigI inhibition and its effects on replication protein dynamics.