Chromosome centromeres, composed of repeated DNA sequences, orchestrate the correct segregation of chromatids in cell division. We have examined the centromeres of human chromosomes 13 and 21 by studying the distribution, in situ, of two alpha satellite sequences that differ in a single nucleotide position. This was possible using padlock probes, oligo-nucleotides that can be ligated into circles upon target recognition. The segregation of individual 13 and 21 homologues in a family was followed by monitoring of the signals from two differentially labelled probes, specific for either sequence variant. A characteristic arrangement of the repeat motifs in three separate spots, oriented transverse to the length axis of the metaphase chromosomes and bilaterally symmetric, indicates that only parts of the detected regions are involved in the centromeric region, joining the sister chromatids before anaphase.
Telomeric length dynamics are thought to play an important role both in the processes of cellular aging and cancer progression. We have revised the primed in situ (PRINS) labeling technique to allow an estimation of the relative length of individual telomeres. We illustrate the applicability of the approach by demonstrating different telomeric sizes not only between blood lymphocytes from a young and an old donor, but also among bone marrow cells from hematological cancer patients. In the latter case we found general variations in telomeric sizes as well as individual telomeric variations that would have escaped detection by other methods. An interesting finding was the selective expansion of a single telomere within a specific subset of cells.
RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51–ssDNA filaments. RECQ5 interacts with RAD51 through protein–protein contacts, and disruption of this interface through a RECQ5–F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51–K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51–I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.
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