Journal: bioRxiv

Article Title: Human POLD3 coordinates leading and lagging strand in Mitotic DNA Synthesis

doi: 10.64898/2025.12.15.694317

Figure Lengend Snippet: a) Structure of human POLD3 with indication of its functional domains and motifs, and of the mu-tations of the various mutant clones we produced. b) Schematic representation of the adapted MiDAS protocol used in this work. c) Results of MiDAS experiments performed on HeLa POLD3 delPIP-box mitotic cells, clone 23 and clone 2C4 versus wild type. Peaks from forward and reverse reads are shown in red and blue, re-spectively. d) Dot plot showing the Index of directionality of the top 15 MiDAS sites (each dot is a MiDAS peak) for the HeLa POLD3 delPIP-box mutant clones and wild type in each of the MiDAS experiments. The p-values reported on the significance bracket come from a Student’s t-test performed between each mutant clone and the wild type control, considering each MiDAS peak as a repeat (statistical significance: p-value<0.05). The Index of Directionality (IoD) for each peak can range between −1 and 1 and is calculated as: An IoD greater than 0 indicates positive directionality (excess of forward reads in the left half peak and/or excess of reverse reads in the right half peak), an IoD smaller than 0 indicates negative direc-tionality (excess of forward reads in the right half peak and/or excess of reverse reads on the left half peak) and IoD equal to 0 indicates no directionality (equal number of forward and reverse reads in both left and right half peaks). For each experiment, a horizontal bar indicates the average IoD value of each cell line. e) Proposed mechanism to explain the directionality observed at MiDAS sites after mutation of the PCNA-interacting motif of POLD3. The delayed production and/or ligation of Okazaki Fragments at MiDAS forks leaves nicks in the lagging strand, making not possible to sequence fragments com-ing from this strand with Next Generation Sequencing. This causes an excess of sequencing reads mapping on the leading strand of the MiDAS forks. f) Average plots of the top 15 peaks for clone 23 (experiment 1), clone 2C4 (experiment 4) and wild type, covering the MiDAS region (from the list provided in ) ±0.1 Mb. The start and end points of the MiDAS peaks were aligned to standardize the different genomic size of the various MiDAS re-gions. Average peaks coming from forward and reverse reads are colored in red and blue, respec-tively. BPM: bins per million mapped reads. BPM (per bin) = number of reads per bin / sum of all reads per bin (in millions).

Article Snippet: After over-night blocking at 4°C in Tris Buffered Saline (TBS) supplemented with 0.1% Tween® 20 (PanReac, AppliChem, Cat. No. A4974,0100) and 5% milk, the membrane was incubated with primary antibodies for POLD3 (rabbit, Bethyl, Cat. No. IHC-00249-T, 1:1000 in TBS-0.1% Tween® 20-5% milk) and PCNA (mouse, Genetex, Cat. No. GTX-20029, 1:5000 in TBS-0.1% Tween® 20-5% milk) for 1 hour at room temperature, washed three times in TBS-0.1% Tween® 20, and incubated with HRP conjugate secondary antibodies anti-rabbit (Anti-Rabbit IgG HRP Conjugate, Promega, Cat. No. W4011) and anti-mouse (Anti-Mouse IgG HRP Conjugate, Promega, Cat. No. W4021) diluted 1:2500 in TBS-0.1% Tween® 20-5% milk for 30 minutes.

Techniques: Functional Assay, Mutagenesis, Clone Assay, Produced, Control, Ligation, Sequencing, Next-Generation Sequencing

Journal: bioRxiv

Article Title: Human POLD3 coordinates leading and lagging strand in Mitotic DNA Synthesis

doi: 10.64898/2025.12.15.694317

Figure Lengend Snippet: Genomic and protein sequence of POLD3 in the various mutant clones used in this work

Article Snippet: After over-night blocking at 4°C in Tris Buffered Saline (TBS) supplemented with 0.1% Tween® 20 (PanReac, AppliChem, Cat. No. A4974,0100) and 5% milk, the membrane was incubated with primary antibodies for POLD3 (rabbit, Bethyl, Cat. No. IHC-00249-T, 1:1000 in TBS-0.1% Tween® 20-5% milk) and PCNA (mouse, Genetex, Cat. No. GTX-20029, 1:5000 in TBS-0.1% Tween® 20-5% milk) for 1 hour at room temperature, washed three times in TBS-0.1% Tween® 20, and incubated with HRP conjugate secondary antibodies anti-rabbit (Anti-Rabbit IgG HRP Conjugate, Promega, Cat. No. W4011) and anti-mouse (Anti-Mouse IgG HRP Conjugate, Promega, Cat. No. W4021) diluted 1:2500 in TBS-0.1% Tween® 20-5% milk for 30 minutes.

Techniques: Sequencing, Mutagenesis, Clone Assay

Journal: bioRxiv

Article Title: Human POLD3 coordinates leading and lagging strand in Mitotic DNA Synthesis

doi: 10.64898/2025.12.15.694317

Figure Lengend Snippet: a) In case of intact dsDNA, both strands are amplified by PCR during library preparation, and ampl-icons coming from both leading and lagging strand are sequenced. b) In case of nicked lagging strand, as it might be the case for the POLD3 delPIP-box mutants, only the leading strand (intact) is exponentially amplified during library preparation and sequenced, while amplicons coming from the lagging strand (nicked) are poorly represented (they are not exponen-tially amplified) and not even sequenced due to their small dimension (small fragments are ex-cluded from sequencing) and to the lack of one of the two adaptor sequences.

Article Snippet: After over-night blocking at 4°C in Tris Buffered Saline (TBS) supplemented with 0.1% Tween® 20 (PanReac, AppliChem, Cat. No. A4974,0100) and 5% milk, the membrane was incubated with primary antibodies for POLD3 (rabbit, Bethyl, Cat. No. IHC-00249-T, 1:1000 in TBS-0.1% Tween® 20-5% milk) and PCNA (mouse, Genetex, Cat. No. GTX-20029, 1:5000 in TBS-0.1% Tween® 20-5% milk) for 1 hour at room temperature, washed three times in TBS-0.1% Tween® 20, and incubated with HRP conjugate secondary antibodies anti-rabbit (Anti-Rabbit IgG HRP Conjugate, Promega, Cat. No. W4011) and anti-mouse (Anti-Mouse IgG HRP Conjugate, Promega, Cat. No. W4021) diluted 1:2500 in TBS-0.1% Tween® 20-5% milk for 30 minutes.

Techniques: Amplification, Sequencing

Journal: bioRxiv

Article Title: Human POLD3 coordinates leading and lagging strand in Mitotic DNA Synthesis

doi: 10.64898/2025.12.15.694317

Figure Lengend Snippet: a) Results of MiDAS experiments performed on HeLa POLD3 delRIR mitotic cells (clone 3D1) and HeLa POLD3 delPP1 mitotic cells (clones 3H2 and 2E4) versus wild type. Peaks from forward and re-verse reads are shown in red and blue, respectively. b) Dot plot showing the Index of directionality of the top 15 MiDAS sites (each dot is a MiDAS peak) for the mutant clones and wild type ineach of the MiDAS experiments. The p-values reported on the significance bracket come from a Student’s t-test performed between each mutant clone and the wild type control, considering each MiDAS peak as a repeat (statistical significance: p-value<0.05). For each experiment, a horizontal bar indicates the average IoD value of each cell line. c) Average plots of the top 15 peaks for clones 3D1, 3H2, 2E4 and wild type, covering the MiDAS region (from the list provided in ) ±0.1 Mb. The start and end points of the MiDAS peaks were aligned to standardize the different genomic size of the various MiDAS regions. Average peaks coming from forward and reverse reads are colored in red and blue, respectively. BPM: bins per million mapped reads. BPM (per bin) = number of reads per bin / sum of all reads per bin (in mil-lions).

Article Snippet: After over-night blocking at 4°C in Tris Buffered Saline (TBS) supplemented with 0.1% Tween® 20 (PanReac, AppliChem, Cat. No. A4974,0100) and 5% milk, the membrane was incubated with primary antibodies for POLD3 (rabbit, Bethyl, Cat. No. IHC-00249-T, 1:1000 in TBS-0.1% Tween® 20-5% milk) and PCNA (mouse, Genetex, Cat. No. GTX-20029, 1:5000 in TBS-0.1% Tween® 20-5% milk) for 1 hour at room temperature, washed three times in TBS-0.1% Tween® 20, and incubated with HRP conjugate secondary antibodies anti-rabbit (Anti-Rabbit IgG HRP Conjugate, Promega, Cat. No. W4011) and anti-mouse (Anti-Mouse IgG HRP Conjugate, Promega, Cat. No. W4021) diluted 1:2500 in TBS-0.1% Tween® 20-5% milk for 30 minutes.

Techniques: Clone Assay, Mutagenesis, Control

Journal: bioRxiv

Article Title: Human POLD3 coordinates leading and lagging strand in Mitotic DNA Synthesis

doi: 10.64898/2025.12.15.694317

Figure Lengend Snippet:

Article Snippet: After over-night blocking at 4°C in Tris Buffered Saline (TBS) supplemented with 0.1% Tween® 20 (PanReac, AppliChem, Cat. No. A4974,0100) and 5% milk, the membrane was incubated with primary antibodies for POLD3 (rabbit, Bethyl, Cat. No. IHC-00249-T, 1:1000 in TBS-0.1% Tween® 20-5% milk) and PCNA (mouse, Genetex, Cat. No. GTX-20029, 1:5000 in TBS-0.1% Tween® 20-5% milk) for 1 hour at room temperature, washed three times in TBS-0.1% Tween® 20, and incubated with HRP conjugate secondary antibodies anti-rabbit (Anti-Rabbit IgG HRP Conjugate, Promega, Cat. No. W4011) and anti-mouse (Anti-Mouse IgG HRP Conjugate, Promega, Cat. No. W4021) diluted 1:2500 in TBS-0.1% Tween® 20-5% milk for 30 minutes.

Techniques: Clone Assay, Mutagenesis

Journal: bioRxiv

Article Title: Human POLD3 coordinates leading and lagging strand in Mitotic DNA Synthesis

doi: 10.64898/2025.12.15.694317

Figure Lengend Snippet: Recent literature suggests that, in MiDAS, DNA polymerase δ might be involved both in leading and lagging strand synthesis, and that the POLD3/PCNA interaction is not involved in the recruit-ment of Polδ at MiDAS sites. Here, we propose that the role of POLD3 in MiDAS might be to co-ordinate leading and lagging strand synthesis. In particular, the C-terminal PIP-box of each of the two POLD3 subunits at the MiDAS fork could bind to the PCNA trimer on the opposite DNA strand, thus providing physical connection between the leading and lagging strand replication ma-chineries.

Article Snippet: After over-night blocking at 4°C in Tris Buffered Saline (TBS) supplemented with 0.1% Tween® 20 (PanReac, AppliChem, Cat. No. A4974,0100) and 5% milk, the membrane was incubated with primary antibodies for POLD3 (rabbit, Bethyl, Cat. No. IHC-00249-T, 1:1000 in TBS-0.1% Tween® 20-5% milk) and PCNA (mouse, Genetex, Cat. No. GTX-20029, 1:5000 in TBS-0.1% Tween® 20-5% milk) for 1 hour at room temperature, washed three times in TBS-0.1% Tween® 20, and incubated with HRP conjugate secondary antibodies anti-rabbit (Anti-Rabbit IgG HRP Conjugate, Promega, Cat. No. W4011) and anti-mouse (Anti-Mouse IgG HRP Conjugate, Promega, Cat. No. W4021) diluted 1:2500 in TBS-0.1% Tween® 20-5% milk for 30 minutes.

Techniques: