Nonhomologous DNA end joining in cell-free systems.

Double strand DNA breaks are usually caused by ionizing radiation and radiomimetic drugs, but can also occur under normal physiological conditions during double strand break-induced recombination, such as the rearrangement of T-cell receptor and immunoglobulin genes during lymphoid development or the mating type switching in yeast. The main repair mechanism for double strand breaks in higher eukaryotes is nonhomologous DNA end joining (NHEJ), which modifies and ligates the two DNA ends without the help of extensive base-pairing interactions for alignment. Defects in double strand break repair are associated with radiosensitivity, predisposition to cancer and immunodeficiency syndromes, and the analysis of the underlying mutations has lead to the identification of several proteins involved in NHEJ. However, these genetic studies have yielded little information on the mechanism of NHEJ, and while some of the protein factors identified possess the expected enzymatic or DNA-binding activities, the precise role of others remains unclear. Systems for cell-free NHEJ have been available for over 10 years, but the biochemical analysis of NHEJ has lagged behind the genetic analysis, and not a single protein factor required for NHEJ has been identified by biochemical purification and reconstitution of NHEJ activity. Here I review the current status of in vitro systems for NHEJ, summarize the results obtained and information gained, and discuss the outlook for biochemical approaches to study NHEJ.

Ku-dependent nonhomologous DNA end joining in Xenopus egg extracts.

An extract from activated Xenopus eggs joins both matching and nonmatching ends of exogenous linear DNA substrates with high efficiency and fidelity (P. Pfeiffer and W. Vielmetter, Nucleic Acids Res. 16:907-924, 1988). In mammalian cells, such nonhomologous end joining (NHEJ) is known to require the Ku heterodimer, a component of DNA-dependent protein kinase. Here I investigated whether Ku is also required for the in vitro reaction in the egg extract. Immunological assays indicate that Ku is very abundant in the extract. I found that all NHEJ was inhibited by autoantibodies against Ku and that NHEJ between certain combinations of DNA ends was also decreased after immunodepletion of Ku from the extract. The formation of a joint between a DNA end with a 5'-protruding single strand (PSS) and an end with a 3'-PSS, between two ends with 3'-PSS, and between two blunt ends was most Ku dependent. On the other hand, NHEJ between two DNA ends bearing 5'-PSS was Ku independent. These results show that the Xenopus cell-free system will be useful to biochemically dissect the role of Ku in eukaryotic NHEJ.

Identification of novel genes encoding transcription elongation factor TFIIS (TCEA) in vertebrates: conservation of three distinct TFIIS isoforms in frog, mouse, and human.

We report the characterization of cDNA clones that define a new, third isoform of the transcription elongation factor TFIIS in Xenopus, mouse, and human. In Xenopus the mRNA of this isoform, termed TFIIS.h, shows tissue-restricted expression, frequently contains unspliced introns, and is characterized by three near-perfect 150-bp repeats at the 5'-terminus. Although we were unable to isolate full-length cDNAs, it is clear that these repeats contain an open reading frame encoding a region of TFIIS.h that is much more complex than in other isoforms. Identification of ESTs encoding TFIIS.h in mouse and human followed by the sequencing of cognate cDNA clones enabled the complete TFIIS.h coding region to be predicted. The conserved N- and C-terminal domains of mammalian TFIIS.h (TCEA3) are separated by a linker region that is more variable in sequence and that is also 50 amino acids longer than in other isoforms. The repetitive region of Xenopus TFIIS.h apparently corresponds to an even more extended linker. Phylogenetic analysis of TFIIS sequences demonstrates the ancient origins of the three vertebrate isoforms, although they appeared functionally equivalent in in vitro RNA cleavage assays.

Transcript cleavage in an RNA polymerase I elongation complex. Evidence for a dissociable activity similar to but distinct from TFIIS.

Stalled Xenopus RNA polymerase I (pol I) elongation complexes bearing a 52-nucleotide RNA were prepared by promoter-initiated transcription in the absence of UTP. When such complexes were isolated and incubated in the presence of Mg2+, the associated RNA was shortened from the 3'-end, and mono- and dinucleotides were released. Shortened transcripts were still associated with the DNA and were quantitatively reelongated upon addition of NTPs. The cleavage activity could be removed from the pol I-ternary complex with buffers containing 0.25% Sarkosyl. These findings indicate that a factor with characteristics similar to elongation factor TFIIS is associated with the pol I elongation complex. However, addition of recombinant Xenopus TFIIS to Sarkosyl-washed pol I elongation complexes had no effect, whereas it showed the expected effects in control reactions with identically prepared pol II elongation complexes. The results thus suggest the existence of a pol I-specific cleavage/elongation factor. I also report the sequence of a novel type of Xenopus TFIIS. The predicted amino acid sequences of the present and previously identified Xenopus TFIIS are less than 65% conserved. Thus, like mammalian species, Xenopus has at least two highly divergent forms of TFIIS.

mRNA encoding the catalytic subunit of DNA-dependent protein kinase is widely expressed in Xenopus cells.

Here, the sequence of a Xenopus laevis cDNA encoding the 640 carboxy-terminal amino acids of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is reported. The predicted Xenopus protein segment is 65% identical to the human counterpart. Northern blot analysis indicates that Xenopus DNA-PKcs is encoded by an approx. 13000 nt transcript. DNA-PKcs mRNA is widely expressed in adult tissues as well as in oocytes and embryos. It is also shown that outside the conserved kinase domain, Xenopus DNA-PKcs bears significant similarities to hypothetical 420.8 and 433.2 kDa proteins in yeast species.

Phosphorylation of the N-terminal domain of Xenopus TATA-box binding protein by DNA-dependent protein kinase depends on the C-terminal core domain.

DNA-dependent protein kinase (DNA-PK) has been shown to phosphorylate several transcription factors in vitro, suggesting that this nuclear enzyme - in addition to its role in DNA repair and recombination - may be involved in transcriptional regulation. In the typical mechanism the DNA-bound kinase phosphorylates a substrate that is bound to the same DNA molecule. Here I report that the Xenopus TATA-box binding protein (xTBP) is hyperphosphorylated by DNA-PK in vitro. The phosphorylation is in the N-terminal domain of the protein but depends fully on the presence of the C-terminal core domain.

Heteroduplex analysis of the Xenopus RNA polymerase I terminator.

Recent results showed that the Xenopus ribosomal terminator, a conserved 9-bp element ("T2/T3 box"), is a pause signal for the RNA polymerase I-elongation complex (Labhart, P. [1995] Nucleic Acids Res. 23, 2252-2258). Since the terminator is known to function only in one orientation, it was of interest to investigate whether the 9-bp pause element had to be present in both DNA-strands to mediate termination of RNA polymerase I-transcription. The present heteroduplex analysis of the terminator shows that only the double-stranded 9-bp element constitutes a functional terminator. Any single-stranded mutation had the same down-effect as the corresponding double-stranded (homoduplex) mutation. Models for termination by Xenopus RNA polymerase I that are supported or eliminated by the present results are discussed.

The Xenopus 9 bp ribosomal terminator (T3 box) is a pause signal for the RNA polymerase I elongation complex.

In Xenopus, termination by RNA polymerase I (pol I) is mediated by the 9 bp sequence GACTTGCNC and RNA 3'-ends are formed -15-20 nt upstream of this terminator element. Here I show that this 9 bp element, also called the 'T3 box', is a pause signal for the elongating transcription complex. The two major transcripts in the paused complex have 3'-ends mapping to 15 and 21 nt upstream of the T3 box, remain bound to the template in 0.25% Sarkosyl, are subject to pyrophosphorolysis and can be chased into longer transcripts. Mutations that reduce overall termination also affect pausing, indicating that pausing is a limiting step in the termination process. Oligonucleotide competition experiments, furthermore, suggest that pausing requires a DNA binding factor. The data support a model in which the first step leading to transcription termination by pol I in Xenopus is pausing of the elongation complex upstream of the T3 box.

DNA-dependent protein kinase specifically represses promoter-directed transcription initiation by RNA polymerase I.

DNA-dependent protein kinase (DNA-PK) is a nuclear enzyme that phosphorylates several transcription factors, but its cellular function has not been elucidated. Here I show that DNA-PK strongly inhibits promoter-directed transcription initiation by Xenopus RNA polymerase I in vitro. The repression is due to protein phosphorylation, since it is relieved by 6-dimethylaminopurine, an inhibitor of protein kinases. DNA-PK inhibits transcription from both linear and circular templates, but the repression is more efficient on linear templates. DNA-PK has no effect on promoter-directed transcription by RNA polymerases II and III. Partial fractionation of the in vitro transcription system shows that a protein fraction containing transcription factor Rib1, the Xenopus equivalent of human SL1, mediates the repression of transcription by DNA-PK. The present data suggest a role for DNA-PK in down-regulating ribosomal gene transcription.

Negative and positive effects of CpG-methylation on Xenopus ribosomal gene transcription in vitro.

Methylation of cytosine-residues in the sequence CpG affects the expression of many genes and generally correlates with reduced transcription. The ribosomal genes of Xenopus laevis were among the first genes to be studied with respect to their DNA methylation, and a loss of methylation during embryonic development correlated with the onset of transcription. Nevertheless, highly methylated ribosomal genes were transcribed at normal levels when injected into oocyte nuclei, and thus transcription of these genes was generally assumed to be insensitive to CpG-methylation. Here I show that Xenopus ribosomal gene transcription can be repressed by cellular factors binding to meCpG, similarly as it has been described for transcription by RNA polymerase II. In the absence of these repressors, however, CpG-methylation has a direct positive effect on RNA polymerase I-promoter activity.

Identification of two steps during Xenopus ribosomal gene transcription that are sensitive to protein phosphorylation.

Protein kinase(s) and protein phosphatase(s) present in a Xenopus S-100 transcription extract strongly influence promoter-dependent transcription by RNA polymerase I. The protein kinase inhibitor 6-dimethyl-aminopurine causes transcription to increase, while the protein phosphatase inhibitor okadaic acid causes transcription to decrease. Repression is also observed with inhibitor 2, and the addition of extra protein phosphatase 1 stimulates transcription, indicating that the endogenous phosphatase is a type 1 enzyme. Partial fractionation of the system, single-round transcription reactions, and kinetic experiments show that two different steps during ribosomal gene transcription are sensitive to protein phosphorylation: okadaic acid affects a step before or during transcription initiation, while 6-dimethylaminopurine stimulates a process "late" in the reaction, possibly reinitiation. The present results are a clear demonstration that transcription by RNA polymerase I can be regulated by protein phosphorylation.

Characterization of two types of ribosomal gene transcription in Xenopus laevis oocytes.

When the germinal vesicle of Xenopus laevis oocytes is translocated into the vegetal hemisphere by centrifugation, the normally silent ribosomal spacer promoters are strongly induced. This induction correlates with the permeability of the nuclear envelope to dextran of molecular weight 70 kDa, thus raising the possibility that the transcriptional changes are due to mixing of nuclear and cytoplasmic components. This basic observation prompted a thorough investigation of ribosomal gene transcription in centrifuged oocytes which had the germinal vesicle either in the animal half (A-oocytes) or in the vegetal half (V-oocytes). Two types of ribosomal gene transcription were characterized: (1) in A-oocytes, spacer promoters remain silent, transcription initiation is dependent on the upstream terminator T3, and transcription is highly processive and recognizes sites of RNA 3' end formation (like T2 and T3); (2) in V-oocytes, spacer promoters are induced, transcription initiation is independent of T3, but most transcripts terminate prematurely after less than 150 nt. Furthermore, the transcription machinery in V-oocytes does not respond to T2 or T3 signals. The implications of the present observations for our understanding of the regulation of the spacer promoters and of the function of the upstream terminator T3 are discussed.

Functional difference between the sites of ribosomal 40S precursor 3' end formation in Xenopus laevis and Xenopus borealis.

In the ribosomal genes of X. laevis, the sequence GACTTGCNC is found about 60bp upstream of the gene promoter (T3) and is necessary and sufficient to cause termination of RNA polymerase I transcription. At the 3' end of the 40S precursor coding region (T2) a sequence differing by one nucleotide, GACTTGCNG, directs RNA 3' end formation but allows polymerase to transcribe on into the intergenic spacer (Labhart and Reeder, 1989, Genes and Dev. 4: 269-276). Sites corresponding to T2 and T3 are also found in a related species, X. borealis. Inspection of the T2 sequence in X. borealis reveals that it contains two copies of the terminator sequence, GACTTGCNC, located 15 and 96 bp downstream of the 3' end of the 40S precursor coding region. Here we present functional tests of those two T2 elements that show that, as predicted from the sequence, they both show termination activity and are functionally indistinguishable from the T3 site in X. laevis. These results suggest that X. laevis T2 is an example of a naturally occurring point mutation, and the inability to terminate transcription at T2 is an exception to the general pattern of ribosomal gene transcription in higher eukaryotes.

A point mutation uncouples RNA 3'-end formation and termination during ribosomal gene transcription in Xenopus laevis.

Two sites, T2 and T3, in the ribosomal gene spacer of Xenopus laevis both direct RNA 3'-end formation 15 bp upstream of the conserved box sequence GACTTGC. Site T2, which defines the 3' end of the 40S precursor, does not terminate transcription whereas site T3 at the 3' end of the spacer does. Here we show that T2 can be converted into a T3-like site with termination activity by a single point mutation 2 bp downstream of the T2 box. RNA 3'-end formation at T2 is unchanged by this mutation. Conversely, a point mutation 2 bp downstream of the T3 box inhibits termination without affecting 3'-end formation. Our results identify two separable events occurring at the 3' end of the ribosomal genes: (1) RNA 3'-end formation by processing and (2) transcription termination. The two processes are directed by two distinct, but overlapping, signals in the DNA sequence. Site T2 in X. laevis is damaged in the second process by a natural mutation.

High initiation rates at the ribosomal gene promoter do not depend upon spacer transcription.

We report experiments that test the model that in Xenopus laevis, RNA polymerase I is "handed over" in a conservative fashion from the T3 terminator to the adjacent gene promoter. We have introduced transcription-terminating lesions into the ribosomal DNA repeat by irradiating cultured cells with ultraviolet light. We used isolated nuclei to measure the effect of such lesions on transcription. UV damage sufficient to prevent all elongating RNA polymerase from reaching T3 from upstream had no adverse effect on the density of RNA polymerase at the very 5' end of the gene. We conclude that high rates of transcription initiation at the gene promoter do not depend upon polymerase passing from one repeat to the next or on polymerase initiating at the spacer promoters.

Ribosomal precursor 3' end formation requires a conserved element upstream of the promoter.

In Xenopus laevis the 3' end of the longest intact ribosomal RNA precursor is formed by a processing event at site T2, which is located 7860 bp downstream of the site of transcription initiation. Processing at T2 is eliminated by mutations within the T2 box, a 7 nucleotide conserved element, GACTTGC, located 15 bp downstream of the 3' ends. The same conserved box is also present at T3, a site 60 bp upstream of the gene promoter and that is part of a termination site. Surprisingly, mutations within the T3 box also eliminate processing at T2. To obtain proper T2 function, T3 can be at any distance but must be in the correct orientation upstream of a ribosomal gene promoter.

A 12-base-pair sequence is an essential element of the ribosomal gene terminator in Xenopus laevis.

rRNA transcription in Xenopus laevis terminates near a 7-base-pair (bp) conserved sequence (T3 box) located 200 bp upstream of the site of transcription initiation for the adjacent gene promoter. We present evidence here that a 12-bp element containing the T3 box is an essential part of the terminator. Using an oocyte injection assay, we found that the 12-bp element (but not the T3 box alone) severely reduced the amount of RNA detectable at sites downstream from itself and that the T3 box within the 12-bp element was required to specify the formation of correct 3' ends. This requirement for the 12-bp element was also seen in pulse-label experiments by using a homogenate of oocyte nuclei, but the present data did not allow us to determine the exact mechanism by which the 12-bp element acts. Removal of the T3 region from its normal location allowed a significant amount of readthrough transcripts to accumulate, indicating that additional sequences may be required for complete terminator function.

Heat shock stabilizes highly unstable transcripts of the Xenopus ribosomal gene spacer.

We have shown recently that, in Xenopus laevis oocytes, the 3' end of the longest detectable ribosomal precursor RNA is not formed by transcription termination but by RNA processing and that RNA polymerase I continues to transcribe through the intergenic spacer region. In oocytes, these spacer transcripts are turned over rapidly, and the only apparent transcription termination site is located 215 base pairs upstream of the 5' end of the next transcription unit. In this paper we show that, at heat shock temperature (34 degrees C), processing at the 3' end of the precursor, rapid turnover of spacer transcripts, and termination are all severely impaired. In contrast, transcription initiation and chain elongation are not significantly affected by heat shock. This results in the appearance of large RNA in the range of 10-20 kilobases and longer.