Fresh off the press:
Langley, A.R., Gräf, S., Smith, J.C. and Krude, T. (2016).
Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq). Nucleic Acids Res 44, 10230–10247.
Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq.
Full publication list:
This link to PubMed opens a new window
Selected recent journal articles:
Kowalski, M.P., Baylis, H.A., and Krude, T. (2015).
Non-coding stem-bulge RNAs are required for cell proliferation and embryonic development in C. elegans. J Cell Sci 128, 2118-2129.
Kowalski, M.P., and Krude, T. (2015).
Functional roles of non-coding Y RNAs. Int J Biochem Cell Biol 66, 20-29.
Wang, I., Kowalski, M.P., Langley, A.R., Rodriguez, R., Balasubramanian, S., Hsu, S.T., and Krude T. (2014).
Nucleotide contributions to the structural integrity and DNA replication initiation activity of noncoding y RNA. Biochemistry 53, 5848-5863.
Collart, C., Christov, C.P., Smith, J.C., and Krude, T. (2011).
The mid-blastula transition defines the onset of Y RNA-dependent DNA replication in Xenopus laevis. Mol Cell Biol 31, 3857-3870.
Zhang, A.T., Langley, A.R., Christov, C.P., Kheir, E., Shafee, T., Gardiner, T.J., and Krude, T. (2011).
Dynamic interaction of Y RNAs with chromatin and initiation proteins during human DNA replication. J Cell Sci 124, 2058-2069.
Krude, T. (2010).
Non-coding RNAs: New players in the field of eukaryotic DNA replication. Subcell Biochem 50, 105-118.
Langley, A.R., Chambers, H., Christov, C.P., and Krude, T. (2010).
Ribonucleoprotein Particles Containing Non-Coding Y RNAs, Ro60, La and Nucleolin Are Not Required for Y RNA Function in DNA Replication. PLoS ONE 5, e13673.
Gardiner, T.J., Christov, C.P., Langley, A.R., and Krude, T. (2009).
A conserved motif of vertebrate Y RNAs essential for chromosomal DNA replication. RNA 15, 1375-1385.
Krude, T., Christov, C.P., Hyrien, O., and Marheineke, K. (2009).
Y RNA functions at the initiation step of mammalian chromosomal DNA replication. J Cell Sci 122, 2836-2845.
Christov, C.P., Trivier, E., and Krude, T. (2008).
Noncoding human Y RNAs are overexpressed in tumours and required for cell proliferation. Br J Cancer 98, 981-988.
Klinge, S., Hirst, J., Maman, J. D., Krude, T., and Pellegrini, L. (2007).
An iron-sulfur domain of the eukaryotic primase is essential for RNA primer synthesis. Nat Struct Mol Biol 14, 875-877.
Christov, C. P., Gardiner, T. J., Szüts, D., and Krude, T. (2006).
Functional requirement of non-coding Y RNAs for human chromosomal DNA replication. Mol Cell Biol 26, 6993-7004.
Krude, T. (2006).
Initiation of chromosomal DNA replication in mammalian cell-free systems. Cell Cycle 5, 2115-2122.
Click here for an open access to the pdf of this review
Nabatiyan, A., Szüts, D., and Krude, T. (2006).
Induction of CAF-1 expression in response to DNA strand breaks in quiescent human cells. Mol Cell Biol 26, 1839-1849.
Marheineke, K., Hyrien, O., and Krude, T. (2005).
Visualization of bidirectional initiation of chromosomal DNA replication in a human cell free system. Nucleic Acids Res 33, 6931-6941.
Szüts, D., Christov, C., Kitching, L., and Krude, T. (2005).
Distinct populations of human PCNA are required for initiation of chromosomal DNA replication and concurrent DNA repair. Exp Cell Res 311, 240-250.
Szüts, D., and Krude, T. (2004).
Cell cycle arrest at the initiation step of human chromosomal DNA replication causes DNA damage. J Cell Sci 117, 4897-4908.
Nabatiyan, A., and Krude, T. (2004).
Silencing of Chromatin Assembly Factor 1 in human cells leads to cell death and loss of chromatin assembly during DNA synthesis. Mol Cell Biol 24, 2853-2862.
Szüts, D., Kitching, L., Christov, C., Budd, A., Peak-Chew, S., and Krude, T. (2003).
RPA is an initiation factor for human chromosomal DNA replication. Nucleic Acids Res 31, 1725-1734.
Keller, C., Hyrien, O., Knippers, R., and Krude, T. (2002).
Site-specific and temporally controlled initiation of DNA replication in a human cell-free system. Nucleic Acids Res 30, 2114-2123.
Laman, H., Coverley, D., Krude, T., Laskey, R., and Jones, N. (2001).
Viral cyclin-cyclin-dependent kinase 6 complexes initiate nuclear DNA replication. Mol Cell Biol 21, 624-635.
Krude, T. (2000).
Initiation of human DNA replication in vitro using nuclei from cells arrested at an initiation-competent state. J Biol Chem 275, 13699-13707.
Krude, T. (1999).
Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner. Exp Cell Res 247, 148-159.
Krude, T., Jackman, M., Pines, J., and Laskey, R.A. (1997).
Cyclin/Cdk-dependent initiation of DNA replication in a human cell-free system.
Cell 88, 109-119.