Spt2p binds DNA and regulates transcription elongation and chromatin structure 
. The synthetic lethality of spt2Δ
with other transcription elongation genes strongly suggests that Spt2p functions in transcription through its sequence non-specific DNA binding activity 
. In addition, the absence of Spt2p caused a loss of histone H3 in transcribed regions and increased recombination between inverted repeats 
. Therefore, Spt2p's sequence non-specific DNA binding activity seems to contribute to genomic integrity, presumably through the regulation of chromatin structure in the transcribed region.
Complete suppression of GCR caused by excess Spt2p by mutations affecting transcription () strongly demonstrates that excess Spt2p might alter transcription and result in GCR formation. The suppression of GCR by these mutations was specific for excess Spt2p-directed GCRs because the fir1Δ
, or cdc73Δ
mutation did not suppress the mre11Δ
mutation-directed GCR formation (data not shown). It should be pointed out that a set1Δ
mutation caused a slight growth defect in the mre11Δ
strain. Transcription synergistically increases the hyper-recombinogenic effect of methyl methane-sulfonate (MMS), suggesting that transcription makes DNA more accessible to genotoxic agents 
. Transcription also introduces topological change that could lead to transient accumulation of ssDNA. The changes in topology and chromatin structure caused by excess Spt2p could produce ssDNA because more RNA polymerase II could occupy the transcribed strands and result in the enlargement of R loops (). In addition, excess Spt2p could bind to the collided junction between the DNA replication fork and transcription that mimics a four-way junction structure through its binding activity to four-way junction structure. The longer un-transcribed ssDNA by excess Spt2p is supported by the high-levels of ssDNA, which produced by excess Spt2p () and the decrease in ssDNA and GCR formation by RNase H, which removes DNA-RNA hybrids (). It has been known that ssDNA is a better substrate for many chemical reactions than double-stranded DNA 
. Long ssDNA can easily be targeted by many modifications including deamination, oxidation as well as simple breaks. Uracil introduced by the deamination of cytosine in ssDNA could be one of the intermediates for GCR formation by excess Spt2p, because expression of human AID that deaminates cytosine increased GCR formation () and Ung1p, an enzyme responsible for removal of Uracil from DNA is required for GCR caused by excess Spt2p (). Similar to what we observed, the hyper-recombination in the hpr1Δ
strain was caused by the increase of DNA-RNA hybrid with the R-loop formation 
A model for GCR formation caused by excess Spt2p.
In addition, large R loops could be mis-recognized as an intermediate in nucleotide excision repair (). When there is DNA damage caused by ultra-violet radiation, nucleotide excision repair proteins denature damaged DNA and create bubble structure. Each end of the bubble is targeted by endonucleases to remove the damaged strand. Yeast Rad1p-Rad10p endonuclease that is homologous to human ERCC1-XPF, makes a nick in the bubble 
. Indeed, ERCC1-XPF could cleave R loops formed in the switch regions during immunoglobulin heavy chain switch recombination in vitro 
. Strong suppression of excess Spt2p-dependent GCR by the rad1Δ
or the rad10Δ
mutation () suggests that large R-loops could be targeted by Rad1p-Rad10p endonuclease. However, we could not detect any significant difference in the level of overall transcription in microarray experiments (data not shown), which could be due to subtle difference in the level of transcription.
Defects in proper DNA replication seem to be a major source of spontaneous GCRs because GCRs accumulate in eukaryotes when S phase checkpoints are abrogated 
. High levels of transcription may cause more collisions between transcription and DNA replication (). Recombination at stalled DNA replication forks increases if there is transcription colliding with it 
. Excess Spt2p increased the population of cells highly in S phase and RNase H could partially reverse this effect (). Therefore, large R loops produced by excess Spt2p could be caused mainly in S phase during DNA replication, presumably due to increased collision between transcription complexes and stalled DNA replication forks. The high increase in GCR in the TEL to CEN strain, containing the highly transcribed TRP1
gene between two negative selection marker genes CAN1
supports the collision model for GCR (). This model is further supported by a GCR increase observed in the GAL-TRP1
strain only when the expression of the TRP1
gene was induced by galactose (). Interestingly, the CEN to TEL strain containing the same high transcription TRP1
gene in a reverse orientation did not cause any significant increase in GCR. It might be due to higher preference of DNA replication in this region of chromosome V from centromeric to telomeric by using ARS507 even though there are multiple late origins at the end of chromosome V. Alternatively, the TEL to CEN strain might have more susceptible chromosome structure for GCR because different orientation of TRP1
gene could produce different chromosome structures.
The Rad5p-Rad18p dependent post-replication repair pathway suppresses GCR formation 
. In contrast to Rad18p-Rad6p that monoubiquitinates proliferating cell nuclear antigen (PCNA) and suppresses GCR formation, Bre1p-Rad6p that monoubiquitinates histone H2B, is required to promote GCR formation in the rad5Δ, rad18Δ,
. In the present study, we found that GCRs produced by excess Spt2p were also suppressed by the rad6Δ
mutation (). GCRs from each individual clone carrying a GCR were all broken chromosomes healed by de novo
telomere addition requiring telomerase and the yKu70-yKu80 heterodimer (). The same type of GCR was observed in rad5Δ, rad18Δ,
. Therefore, it is possible that certain types of GCR could be preferentially generated when DNA damage at stalled forks collide with transcription complexes. Further investigations are necessary to elucidate mechanisms. Intriguingly, Rad5p has a Swi2/Snf2 domain that has been suggested to function in altering chromatin structure. Although there is no direct evidence that yeast Rad5p functions in transcription, it might modulate transcription of genes near the stalled DNA replication forks.
The HMG1 protein is a non-histone DNA binding protein and regulates the transcription of many genes through its interaction with other proteins involved in transcription. Transcription profiling showed a dramatic increase of HMG1 expression in more than 80% of gastric cancers 
. In addition, various cancer cells including melanoma expressed higher levels of HMG1 protein 
. Therefore, high levels of HMG1 protein seem to be closely linked to carcinogenesis. Previous studies of HMG1 overexpression in cancers mainly revealed its role in activating transcription of certain genes such as the Melanoma Inhibitory Activity (MIA) for the progression of carcinogenesis 
. Our novel discovery demonstrating the high enhancement of GCR formation by an HMG-like protein suggests that Spt2p can add new mechanistic detail to carcinogenesis linked to transcription imbalance and genomic integrity.