Abstract
Homologous recombination (HR) is an important DNA repair pathway and is essential for cell survival. It plays an important role in the repair of replication-associated injuries and is functionally connected to replication. Transcription is another cellular process that has emerged to have a connection to human resources. Transcription enhances HR, which is a pervasive phenomenon called transcription recombination (TR).
Recent evidence suggests that TAR plays a role in inducing genetic instability, for example in THO mutants (Tho2, Hpr1, Mft1 and Thp2) in yeast or during immune system development leading to genetic diversity in yeast. the mammals. On the other hand, evidence also suggests that TAR may play a role in preventing genetic instability in many different ways, one of which is by rescuing replication during transcription.
Therefore, TAR is a double-edged sword and plays a role in both preventing and inducing genetic instability. Despite the interesting nature of TAR, the mechanism behind TAR remains elusive. Recent advances in the area, however, suggest a link between TAR and replication and show specific genetic requirements for TAR that differ from normal HR. In this review, we aim to present the available evidence for TAR in higher and lower eukaryotes and discuss its possible mechanisms, with an emphasis on its connection to replication.
Material and methods
- Gene expression profiling experiments
Total RNA was isolated from isogenic MLL-/- and MLL+/+ control cell lines on three independent days, mixed and used for hybridization of Mouse Genome 430 2.0 arrays. All experiments were replicated twice to identify upregulated and downregulated genes. The gene list was created using only significantly (Δ > 2-fold) modified genetic signatures. All Affymetrix experiments were performed on the NGFN II Affymetrix work platform in Essen (L Klein-Hitpass). Comparison analysis was performed using the FileMaker database program. Complete lists of gene expression profiles are available upon request.
- Cloning and Transient Transfection of Internal Human and Mouse Gene Promoter Elements
Plasmid pGL3-Ah (−492/ex12/+768) was generated by PCR using the oligonucleotides Prom·3-XhoI (5′-CCGCTCGAG CAGTGAGTCGAGATCGCACC-3′) and Prom·5-HindIII (5′-CCCAAGCTTCCCAAAATTAAAACACAAAATAGG-3′). ). An arbitrary splice acceptor site (5′-AAGCTTTTTGCCATTATATTTTCTTACAGCAGCAAGCTT-3′) was cloned into a unique HindIII restriction site to ensure splice between exon 12 and the luciferase reporter gene.
Plasmids pGL3-Bh (−35/ex12/+768) and pGL3-Ch (−492/ex12Δ) were constructed accordingly using oligonucleotides Prom2 3-XhoI (5′-ccgctcgagTGACATACTTCTATCTTCCCAT-3′ and Prom3 5-Hind ( 5′-cccaagcttAAGGGCTCAACACAGACTT GG-3′) A similar strategy was applied to obtain the 3 pGL3-A/B/Cm mouse promoter constructs, except that the natural splice site of exon 13 of Mll was fused in frame with the Luciferase open reading frame All constructs were transiently co-transfected into 4 × 10 4 HeLa cells using calcium phosphate DNA pellets of 1 µg luciferase and 20 ng pSEAP plasmid (internal control).
- Experiments with genetic reporters
Gene reporter experiments were performed using pGL3-Luciferase plasmids as described (Uhl et al., 2002). The promoterless pGL3-Basic vector was used as a negative control, while an SV40 promoter::luciferase expression constructs served as a positive control (pGL3-SV40). All experiments were performed independently three times in triplicate.
- Western blot experiments
(107) Cells were lysed for 20 min at 4 °C in buffer A (20 mM Hepes pH 7.7, 150 mM NaCl, 1 % Triton X-100 and 0.4 mM ethylenediamine-N,N,N′, N′-tetraacetic acid (EDTA)). After centrifugation (20 min, 14,000 × g, 4°C), the supernatant (S150) was stored. The nuclei pellet was incubated for 20 min at 4°C in buffer B (20 mM Hepes pH 7.7, 450 mM NaCl, 1% Triton X-100 and 0.4 mM EDTA). After centrifugation (20 min, 14,000 × g, 4 °C), the supernatant (S450) was collected.
Immunoprecipitations with polyclonal antiserum 173 (PAS173) were performed from whole cell lysates prepared from 107 or 2 × 107 cells with lysis buffer A. The supernatant was used for paging analysis with sodium dodecyl sulfate. (SDS) and Western blot experiments. Western blots were tested with MAB E28 (raised against human MLL protein sequence 2328-2660) or PAS173 specific for human MLL·C protein fragment (Nakamura et al., 2002).