Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Bloom Syndrome Helicase Stimulates RAD51 DNA Strand Exchange Activity through a Novel Mechanism

Loss or inactivation of BLM, a helicase of the RecQ family, causes Bloom syndrome, a genetic disorder with a strong predisposition to cancer. Although the precise function of BLM remains unknown, genetic data has implicated BLM in the process of genetic recombination and DNA repair. Previously, we demonstrated that BLM can disrupt the RAD51-single-stranded DNA filament that promotes the initial steps of homologous recombination. However, this disruption occurs only if RAD51 is present in an inactive ADP-bound form. Here, we investigate interactions of BLM with the active ATP-bound form of the RAD51-single-stranded DNA filament. Surprisingly, we found that BLM stimulates DNA strand exchange activity of RAD51. In contrast to the helicase activity of BLM, this stimulation does not require ATP hydrolysis. These data suggest a novel BLM function that is stimulation of the RAD51 DNA pairing. Our results demonstrate the important role of the RAD51 nucleoprotein filament conformation in stimulation of DNA pairing by BLM.Mutations of BLM helicase cause Bloom syndrome (BS),² a rare autosomal disorder, which is associated with stunted growth, facial sun sensitivity, immunodeficiency, fertility defects, and a greatly elevated incidence of many types of cancer occurring at an early age (1). BLM belongs to the highly conserved family of RecQ helicases that are required for the maintenance of genome integrity in all organisms (2, 3). There are five RecQ helicases in humans; mutations in three of them, WRN, RECQ4, and BLM, have been associated with the genetic abnormalities known as Werner, Rothmund-Thomson, and Bloom syndrome, respectively (4, 5). The cells from BS patients display genomic instability; the hallmark of BS is an increase in the frequency of sister chromatid and interhomolog exchanges (1, 6). Because homologous recombination (HR) is responsible for chromosomal exchanges, it is thought that BLM helicase functions in regulating HR (7–9). Also, BLM helicase is required for faithful chromosome segregation (10) and repair of stalled replication forks (11, 12), the processes that are linked to HR (13–15). BLM was found to interact physically with RAD51, a key protein of HR (16) that catalyzes the central steps in HR including the search for homology and the exchange of strands between homologous ssDNA and dsDNA sequences (17). In cells, BLM forms nuclear foci, a subset of which co-localize with RAD51. Interestingly, the extent of RAD51 and BLM co-localization increases in response to ionizing radiation, indicating a possible role of BLM in the repair of DNA double-strand breaks (16).Biochemical studies suggest that BLM may perform several different functions in HR. BLM was shown to promote the dissociation of HR intermediates (D-loops) (18–20), branch migration of Holliday junctions (21), and dissolution of double Holliday junctions acting in a complex with TopoIIIα and BLAP75 (22–24). BLM may also facilitate DNA synthesis during the repair process by unwinding the DNA template in front of the replication fork (25). In addition, BLM and its yeast homolog Sgs1 may play a role at the initial steps of DNA double-strand break repair by participating in exonucleolitic resection of the DNA ends to generate DNA molecules with the 3′-ssDNA tails, a substrate for RAD51 binding (26–29).In vivo, the process of HR is tightly regulated by various mechanisms (30). Whereas some proteins promote HR (14, 31), others inhibit this process, thereby preventing its untimely initiation (32, 33). Disruption of the Rad51-ssDNA nucleoprotein filament appears to be an especially important mechanism of controlling HR. This filament disruption activity was demonstrated for the yeast Srs2 helicase (34, 35) and human RECQ5 helicase (36). Recently, we found that BLM can also catalyze disruption of the RAD51-ssDNA filament (25). This disruption only occurs if the filament is present in an inactive ADP-bound form, e.g. in the presence of Mg²⁺. Conversion of RAD51 into an active ATP-bound form, e.g. in the presence of Ca²⁺ (37), renders the filament resistant to BLM disruption (25). In this study, we analyze the interactions of BLM with an active ATP-bound RAD51-ssDNA filament. Surprisingly, we found that BLM stimulates the DNA strand exchange activity of RAD51. Thus, depending on the conformational state of the RAD51 nucleoprotein filament, BLM may either inhibit or stimulate the DNA strand exchange activity of RAD51. Our analysis demonstrated that, in contrast to several known stimulatory proteins that act by promoting formation of the RAD51-ssDNA filament, BLM stimulates the DNA strand exchange activity of RAD51 at a later stage, during synapsis. Stimulation appears to be independent of the ATPase activity of BLM. We suggest that this stimulation of RAD51 may represent a novel function of BLM in homologous recombination.     

PTIP Regulates 53BP1 and SMC1 at the DNA Damage Sites

Although PTIP is implicated in the DNA damage response, through interactions with 53BP1, the function of PTIP in the DNA damage response remain elusive. Here, we show that RNF8 controls DNA damage-induced nuclear foci formation of PTIP, which in turn regulates 53BP1 localization to the DNA damage sites. In addition, SMC1, a substrate of ATM, could not be phosphorylated at the DNA damage sites in the absence of PTIP. The PTIP-dependent pathway is important for DNA double strand breaks repair and DNA damage-induced intra-S phase checkpoint activation. Taken together, these results suggest that the role of PTIP in the DNA damage response is downstream of RNF8 and upstream of 53BP1. Thus, PTIP regulates 53BP1-dependent signaling pathway following DNA damage.The DNA damage response pathways are signal transduction pathways with DNA damage sensors, mediators, and effectors, which are essential for maintaining genomic stability (1–3). Following DNA double strand breaks, histone H2AX at the DNA damage sites is rapidly phosphorylated by ATM/ATR/DNAPK (4–10), a family homologous to phosphoinositide 3-kinases (11, 12). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and the MRE11·RAD50·NBS1 complex (13, 14), at the DNA damage sites. Translocalization of these proteins to the DNA double strand breaks (DSBs)³ facilitates DNA damage checkpoint activation and enhances the efficiency of DNA damage repair (14, 15).Recently, PTIP (Pax2 transactivation domain-interacting protein, or Paxip) has been identified as a DNA damage response protein and is required for cell survival when exposed to ionizing radiation (IR) (1, 16–18). PTIP is a 1069-amino acid nuclear protein and has been originally identified in a yeast two-hybrid screening as a partner of Pax2 (19). Genetic deletion of the PTIP gene in mice leads to early embryonic lethality at embryonic day 8.5, suggesting that PTIP is essential for early embryonic development (20). Structurally, PTIP contains six tandem BRCT (BRCA1 carboxyl-terminal) domains (16–18, 21). The BRCT domain is a phospho-group binding domain that mediates protein-protein interactions (17, 22, 23). Interestingly, the BRCT domain has been found in a large number of proteins involved in the cellular response to DNA damages, such as BRCA1, MDC1, and 53BP1 (7, 24–29). Like other BRCT domain-containing proteins, upon exposure to IR, PTIP forms nuclear foci at the DSBs, which is dependent on its BRCT domains (16–18). By protein affinity purification, PTIP has been found in two large complexes. One includes the histone H3K4 methyltransferase ALR and its associated cofactors, the other contains DNA damage response proteins, including 53BP1 and SMC1 (30, 31). Further experiments have revealed that DNA damage enhances the interaction between PTIP and 53BP1 (18, 31).To elucidate the DNA damage response pathways, we have examined the upstream and downstream partners of PTIP. Here, we report that PTIP is downstream of RNF8 and upstream of 53BP1 in response to DNA damage. Moreover, PTIP and 53BP1 are required for the phospho-ATM association with the chromatin, which phosphorylates SMC1 at the DSBs. This PTIP-dependent pathway is involved in DSBs repair.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Destabilization of Yeast Micro- and Minisatellite DNA Sequences by Mutations Affecting a Nuclease Involved in Okazaki Fragment Processing (rad27) and DNA Polymerase δ (pol3-t)

We examined the effects of mutations in the Saccharomyces cerevisiae RAD27 (encoding a nuclease involved in the processing of Okazaki fragments) and POL3 (encoding DNA polymerase δ) genes on the stability of a minisatellite sequence (20-bp repeats) and microsatellites (1- to 8-bp repeat units). Both the rad27 and pol3-t mutations destabilized both classes of repeats, although the types of tract alterations observed in the two mutant strains were different. The tract alterations observed in rad27 strains were primarily additions, and those observed in pol3-t strains were primarily deletions. Measurements of the rates of repetitive tract alterations in strains with both rad27 and pol3-t indicated that the stimulation of microsatellite instability by rad27 was reduced by the effects of the pol3-t mutation. We also found that rad27 and pol3-01 (an allele carrying a mutation in the “proofreading” exonuclease domain of DNA polymerase δ) mutations were synthetically lethal.All eukaryotic genomes thus far examined contain many simple repetitive DNA sequences, tracts of DNA with one or a small number of bases repeated multiple times (48). These repetitive regions can be classified as microsatellites (small repeat units in tandem arrays 10 to 60 bp in length) and minisatellites (larger repeat units in tandem arrays several hundred base pairs to several kilobase pairs in length). In this paper, arrays with repeat units 14 bp or less will be considered microsatellites and arrays with longer repeat units will be considered minisatellites.Previous studies show that simple repetitive sequences are unstable relative to “normal” DNA sequences, frequently undergoing additions or deletions of repeat units, in Escherichia coli (24), Saccharomyces cerevisiae (12), and mammals (59). This mutability has two important consequences. First, it results in polymorphic loci that are useful in genetic mapping and forensic studies (15, 59). Second, although these repetitive tracts are usually located outside of coding sequences, alterations in the lengths of microsatellites or minisatellites located within coding sequences can produce frameshift mutations or novel protein variants (20, 22, 26).From studies of the effects of various mutations on microsatellite stability in yeast and E. coli (40) and the analysis of mutational changes caused by DNA polymerase in vitro (21), it is likely that most alterations reflect DNA polymerase slippage events (47). These events involve the transient dissociation of the primer and template strands during the replication of a microsatellite (Fig. ​(Fig.1).1). If the strands reassociate to yield an unpaired repeat on the primer strand, the net result is an addition of repeats (following a second round of DNA replication). Unpaired repeats on the template strand would result in a deletion by the same mechanism. Open in a separate windowFIG. 1“Classical” model for the generation of microsatellite alterations by DNA polymerase slippage. Two single strands of a replicating DNA molecule are shown, with each repeat unit indicated by a rectangle. Arrows indicate the 3′ ends of the strand, and the top and bottom strands represent the elongating primer strand and the template strand, respectively. Step 1, the primer and template strand dissociate; step 2, the primer and template strands reassociate in a misaligned configuration, resulting in an unpaired repeat on either the template strand (left side) or primer strand (right side); step 3, DNA synthesis is completed. If the unpaired repeats are not excised by the DNA mismatch repair system, after the next round of DNA synthesis one DNA molecule will be shortened by one repeat (left side) or lengthened by one repeat (right side).A number of mutations have been shown to elevate microsatellite instability. In E. coli (24, 46), yeast (44, 45), and mammalian cells (27), mutations in genes affecting DNA mismatch repair dramatically elevate the instability of a dinucleotide microsatellite. The most likely explanation of this result is that the DNA mismatches (unpaired repeats) resulting from DNA polymerase slippage events are efficiently removed from the newly synthesized strand by the DNA mismatch repair system. Thus, in the absence of mismatch repair, tract instability is elevated. From genetic studies, it has been found that mismatch repair in yeast efficiently corrects DNA mismatches involving 1- to 14-base loops (the size of the repeat units in microsatellites) but fails to correct mismatches involving loops larger than 16 bases (the size of the repeat units in minisatellites) (3, 41, 53). An inefficient mechanism, not involving the classical DNA mismatch repair system, is capable of correcting large DNA loops formed during meiotic recombination (19).In addition to mutations affecting DNA mismatch repair, some mutations affecting DNA replication in yeast destabilize microsatellites. Yeast strains bearing a null mutation in the RAD27 (RTH1) gene have high levels of instability of the dinucleotide poly(GT) and the trinucleotide CAG, specifically elevating single-repeat insertions (18, 39). RAD27 encodes the homolog of the mammalian FEN-1 protein, a 5′-to-3′ exonuclease (10, 11, 33). This nuclease activity is required for removing the terminal ribonucleotide residue from the 5′ end of the Okazaki fragment (9, 14, 35, 54, 55, 57); this step is necessary for the two adjoining fragments to be ligated together. FEN-1 appears to be active as either an exonuclease in the presence of a single-stranded gap upstream of the 5′ terminus or an endonuclease on a 5′ flap structure (13, 34). Since yeast strains that contain a null mutation in RAD27 grow poorly but are viable (38, 43), it is likely that less efficient nuclease activities that are also capable of 5′ Okazaki fragment processing are present in yeast. In addition to destabilizing dinucleotide microsatellites, rad27 strains have high levels of spontaneous mitotic recombination, elevated rates of forward mutation, and increased sensitivity to the alkylating agent methyl methanesulfonate (MMS) (18, 38, 43). In contrast to the mutations normally seen in mismatch repair mutants, i.e., point mutations or small frameshifts, the types of mutations observed in the absence of Rad27p are duplications of sequences flanked by short direct repeats (4 to 7 bp in length) (49). These duplications were not affected by the DNA mismatch repair system.The same class of sequences that are duplicated in the rad27 strains show an elevated rate (up to 1,000-fold) of deletion in strains containing a temperature-sensitive allele (pol3-t) of the yeast gene encoding DNA polymerase δ (52, 53). This mutant (initially named tex1) was isolated in a strain that exhibited an increased excision rate of a bacterial transposon with long terminal repeats inserted within a yeast gene (7). The pol3-t allele, which encodes a mutation (Gly₆₄₁ to Ala₆₄₁) (51) located near the putative nucleotide binding and active-site domains of the enzyme (58), is thought to diminish the rate of lagging-strand synthesis resulting in long stretches of single-stranded DNA on the lagging-strand template (8). This single-stranded DNA may have the potential to form intrastrand base-paired structures, creating interactions between short direct repeats. These interactions would result in an increased frequency of deletions caused by DNA polymerase slippage.Since rad27 and pol3-t mutations elevate the rates of duplications and deletions associated with short separated repeats in nonrepetitive DNA sequences, Kunkel et al. (22) suggested that these mutations could also destabilize minisatellites. In this paper, we examine the effects of rad27 and pol3-t mutations on the stability of simple repeats in which the repeat unit length varies between 1 and 20 bp. Our results show that both mutations destabilize both microsatellites and minisatellites, but that the mechanisms involved in the destabilization are different for the two mutations.