LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells.

Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype.

SPO11-independent DNA repair foci and their role in meiotic silencing.

In mammalian meiotic prophase, the initial steps in repair of SPO11-induced DNA double-strand breaks (DSBs) are required to obtain stable homologous chromosome pairing and synapsis. The X and Y chromosomes pair and synapse only in the short pseudo-autosomal regions. The rest of the chromatin of the sex chromosomes remain unsynapsed, contains persistent meiotic DSBs, and the whole so-called XY body undergoes meiotic sex chromosome inactivation (MSCI). A more general mechanism, named meiotic silencing of unsynapsed chromatin (MSUC), is activated when autosomes fail to synapse. In the absence of SPO11, many chromosomal regions remain unsynapsed, but MSUC takes place only on part of the unsynapsed chromatin. We asked if spontaneous DSBs occur in meiocytes that lack a functional SPO11 protein, and if these might be involved in targeting the MSUC response to part of the unsynapsed chromatin. We generated mice carrying a point mutation that disrupts the predicted catalytic site of SPO11 (Spo11(YF/YF)), and blocks its DSB-inducing activity. Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11(YF/YF) and Spo11 knockout meiocytes. These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body. In SPO11-deficient oocytes, the number of repair foci increased during oocyte development, indicating the induction of S phase-independent, de novo DNA damage. In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes. Taken together, our results indicate that in addition to asynapsis, persistent SPO11-induced DSBs are important for the initiation of MSCI and MSUC, and that SPO11-independent DNA repair foci contribute to the MSUC response in oocytes.

The retinoblastoma gene family in cell cycle regulation and suppression of tumorigenesis.

Since its discovery in 1986, as the first tumor suppressor gene, the retinoblastoma gene (Rb) has been extensively studied. Numerous biochemical and genetic studies have elucidated in great detail the function of the Rb gene and placed it at the heart of the molecular machinery controlling the cell cycle. As more insight was gained into the genetic events required for oncogenic transformation, it became clear that the retinoblastoma gene is connected to biochemical pathways that are dysfunctional in virtually all tumor types. Besides regulating the E2F transcription factors, pRb is involved in numerous biological processes such as apoptosis, DNA repair, chromatin modification, and differentiation. Further complexity was added to the system with the discovery of p107 and p130, two close homologs of Rb. Although the three family members share similar functions, it is becoming clear that these proteins also have unique functions in differentiation and regulation of transcription. In contrast to Rb, p107 and p130 are rarely found inactivated in human tumors. Yet, evidence is accumulating that these proteins are part of a "tumor-surveillance" mechanism and can suppress tumorigenesis. Here we provide an overview of the knowledge obtained from studies involving the retinoblastoma gene family with particular focus on its role in suppressing tumorigenesis.