Paulina Prorok

Institut de Génétique Humaine · Replication and Genome Dynamics

The accuracy of replication is one of the most important mechanisms ensuring the stability of the genome. The fork protection complex prevents premature replisome stalling and/or premature disassembly upon stress. Here, we characterize the Timeless–Tipin complex, a component of the fork protection complex. We used microscopy approaches, including c...

DNA replication is a fundamental process ensuring the maintenance of the genome each time cells divide. This is particularly relevant early in development when cells divide profusely, later giving rise to entire organs. Here, we analyze and compare the genome replication progression in human embryonic stem cells, induced pluripotent stem cells, and...

Various origin mapping approaches have enabled genome-wide identification of origins of replication (ORI) in model organisms, but only a few studies have focused on divergent organisms. By employing three complementary approaches we provide a high-resolution map of ORIs in Plasmodium falciparum, the deadliest human malaria parasite. We profiled the...

Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interph...

Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interph...

Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interph...

Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interph...

In metazoan cells, DNA replication initiates from thousands of genomic loci scattered throughout the genome called DNA replication origins. Origins are strongly associated with euchromatin, particularly open genomic regions such as promoters and enhancers. However, over a third of transcriptionally silent genes are associated with DNA replication i...

Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interph...

It was proposed that the last universal common ancestor (LUCA) evolved under high temperatures in an oxygen-free environment, similar to those found in deep-sea vents and on volcanic slopes. Therefore, spontaneous DNA decay, such as base loss and cytosine deamination, was the major factor affecting LUCA’s genome integrity. Cosmic radiation due to E...

To ensure error-free duplication of all (epi)genetic information once per cell cycle, DNA replication follows a cell type and developmental stage specific spatio-temporal program. Here, we analyze the spatio-temporal DNA replication progression in (un)differentiated mouse embryonic stem (mES) cells. Whereas telomeres replicate throughout S-phase, w...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Aerobic respiration generates reactive oxygen species (ROS), which can damage nucleic acids, proteins and lipids. A number of transcription factors (TFs) contain redox-sensitive cysteine residues at their DNA-binding sites, hence ROS-induced thiol oxidation strongly inhibits their recognition of the cognate DNA sequences. Major human apurinic/apyri...

Genome-wide studies of DNA replication origins revealed that origins preferentially associate with an Origin G-rich Repeated Element (OGRE), potentially forming G-quadruplexes (G4). Here, we functionally address their requirements for DNA replication initiation in a series of independent approaches. Deletion of the OGRE/G4 sequence strongly decreas...

DNA replication starts with the opening of DNA at sites called DNA replication origins. From the single sequence-specific DNA replication origin of the small Escherichia coli genome, up to thousands of origins that are necessary to replicate the large human genome, strict sequence specificity has been lost. Nevertheless, genome-wide analyses perfor...

Among other targets, the protein lysine methyltransferase PR-Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4-20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication...

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific D...

Apurinic/apyrimidinic (AP) endonucleases are key DNA repair enzymes involved in the base excision repair (BER) pathway. In BER, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases and/or oxidative damage. A Triticum aestivum cDNA encoding for a putative homologue of ExoIII family AP endonucleases which...

Significance Hydrolytic deamination of cytosine to uracil generates a highly mutagenic DNA base lesion and is considered one of the major sources of spontaneous mutation in living organisms. We report that the major human apurinic/apyrimidinic (AP) endonuclease, APE1, is a deoxyuridine endonuclease and can remove uracil residues in the DNA glycosyl...

Oxygen free radicals induce lipid peroxidation (LPO) that damages and breaks polyunsaturated fatty acids in cell membranes. LPO-derived aldehydes and hydroxyalkenals react with DNA leading to the formation of etheno(ε)-bases including 1,N(6)-ethenoadenine (εA) and 3,N(4)-ethenocytosine (εC). The εA and εC residues are highly mutagenic in mammalian...

Action of various NIR AP endonucleases towards oligonucleotide duplexes containing a single ε-base. A solution of 10 nM of 22 mer 3′-[32P]-labelled εA22•T, εC22•G αdA-RT•T and Tg-RT•A oligonucleotide duplexes was incubated with either Nfo, or Apn,1 or 10 nM APE1 for 30 min and 2 h at 37°C. (A) Lane 1, control, non-treated εA22•T; lane 2, as 1 but 2...

In vitro reconstitution of the long-patch NIR pathway using oligonucleotide duplex containing single αdA residue. 10 nM of non-labelled 34 mer αA-PN•T oligonucleotide duplex was incubated for 3 h at 37°C in the presence of 10 nM APE1, 2 nM FEN1, 0.02 U POLβ and 20 U T4 DNA ligase in the reaction buffer containing 50 mM HEPES-KOH (pH 7.2), 30 mM NaC...

Comparison of NIR and AP endonuclease activities of APE1-WT and mutant APE1-D308A proteins. A solution of 10 nM of 22 mer 3′-[32P]-labelled εA22•T, THF•T and αdA•T oligonucleotide duplexes were incubated with varying amounts of the APE1 proteins under NIR conditions, and products of the reaction were analyzed using denaturing PAGE. (A) εA22•T duple...

Time kinetics of in vitro reconstitution of the NIR and BER pathways using oligonucleotide duplex containing single αdA or εA residue. (A,B) A solution of 10 nM of non-labelled oligonucleotide duplexes was incubated at 37°C for various times up to 120 min in the presence of 10 nM APE1, 2 nM FEN1, 0.01 U POLβ and 4 U T4 DNA ligase in a reaction buff...

Dependence of APE1-catalyzed NIR activity on reaction conditions. A solution of 10 nM of 3′-[32P]-labelled oligonucleotide duplex containing a single ε-base was incubated for 1–2 h at 37°C with 5 nM APE1 under NIR and BER conditions. (A) εC22•G and (B) εA22•T duplexes. Lanes 1 and 9, control, non-treated duplex; lanes 2 and 10, duplex incubated wit...

Electrophoretic Mobility Shift Assay (EMSA) for binding of APE1 to oligonucleotide duplexes containing a single ε-base. The standard binding reaction mixture (20 µl) contained 20 mM Hepes-KOH, pH 7.6, 50 mM KCl, 10 µM or 100 µM MgCl2, 10 nM of 22 mer 3′-[32P]-labelled εA22•T, A22•T, εC22•G or C22•G and 250 nM or 500 nM APE1. The mixture was incubat...

SPRi kinetic curves of APE1 (37–296 nM) interacting with immobilized DNA on the pre-treated surface. (A–C) Measurements were performed in “BER+EDTA” buffer. (D–F) Measurements were performed in NIR buffer. The curves representing the interactions of APE1 with Hairpin DNA (HP) are in blue, regular single-stranded T22 oligonucleotides are in grey, εA...

Kinetic constants for APE1-DNA substrate interactions deduced from sensorgrams using Bia evaluation software. (DOC)

One of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to G>C≫A>T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we tested survival and mutation spectra in the lacZα gene of M13mp18 phage, whose DNA was treated in vitro...

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision...