A) The two-hour induction of Spt2p enhances the GCR rate as compared to the control that carried the plasmid backbone only. B) The GCR produced by excess Spt2p was significantly reduced by the mutation in yKU80, RAD10, RAD1, or TLC1 genes. C) Western blot analysis of FLAG tagged Spt2p demonstrates that yku80, rad10, rad1, or tlc1 mutation did not cause significant change in expression levels of Spt2p. The intensity of each band from Spt2p was divided by the intensity of band from tubulin control. The induction value was calculated by dividing the number after the galactose induction (+) with the number before the galactose induction (−). The fold difference with wild type was calculated by setting the induction value of wild type to 1.

A) Spt2p has four different domains important for DNA binding. The C-terminal domain has a lysine residue (italic letter). The K325A point mutation is located near the C-terminus of Spt2p where there are large numbers of positive charged amino acids that are underlined in the figure. B) Overexpression of different domains of Spt2p enhanced GCR formation. C) The K325A point mutation completely blocked the increased GCR produced by excess Spt2p. D) Western blot analysis confirmed that the K325A point mutation did not affect the expression or the stability of Spt2p. α represents antibody. β-tubulin1 was used as a control. The fold difference with wild type was calculated as described in Figure 1 legend.

A) Inactivation of Bur2p, Rad6p, or Bre1p blocked GCR produced by excess Spt2p. B) Mutation in either SET1 or DOT1 reduced the GCR formation by excess Spt2p. C) Proteins participating in transcription elongation including Cdc73p in PAF complex, Hir1p, Swr1p, and Dst1p are required for GCR caused by excess Spt2p. D) Mutation in HPR1 or FIR1 abolished GCR caused by excess Spt2p. The bottom panel of each section shows the expression level of Spt2p in strain backgrounds used in the study. α represents antibody. β-tubulin was used as a control. The fold difference with wild type was calculated as described in Figure 1 legend.

A) Enhanced GCR formation by excess Spt2p was partially alleviated by RNase H expression. B) Excess Spt2p expression increased the amount of ssDNA in cells. DNA equally loaded on two membranes was subjected to hybridization with radio-labeled DNA that was PCR amplified from yeast chromosome V (31121–31859) that covers part of two ORFs, AVT2 and CAN1. The denatured condition measured the quantity of DNA loaded whereas its native condition measured the amount of ssDNA. C) FACS analysis of cells carrying control plasmids, carrying Spt2p overexpression (o/e) only, and carrying both Spt2p and RNase H overexpression plasmids shows that excess Spt2p causes more cells to stay in S phase and RNase H expression decreases the S phase population.

A) Expression of human AID in yeast increased the GCR rate. B) Mutation in UNG1 abolishes GCR caused by excess Spt2p.

A) Treatment of 6-AU suppresses GCR caused by excess Spt2p. B) Chromosome V structures of two yeast strains; CEN to TEL chromosome V has the TEF-TRP1 gene inserted between the URA3 and CAN1 genes from centromeric to telomeric direction. TEL to CEN chromosome V carries the TEF-TRP1 gene in the same location in the opposite direction. C) TEL to CEN strain enhances GCR formation. The GCR fold induction of each strain with or without 0.1% MMS treatment is demonstrated. WT represents a strain having no TEF-TRP1 gene. D) Excess Spt2p enhances further in GCR formation in TEL to CEN strain. GCR fold inductions from experiments were calculated by setting the GCR frequency of WT strain without MMS treatment as 1. – and + represents without and with treatment of MMS, respectively. Ctrl represents no Spt2p overexpression. E) Chromosome V structure of strain having Galactose inducible TRP1 gene (GAL-TRP1) between marker genes, CAN1 and URA3 for GCR assay. F) Galactose driven transcription of TRP1 gene enhanced GCR formation. WT represents a strain having no GAL-TRP1 gene. Glu and Gal represent glucose and galactose supplied in media, respectively.

A) DNA replication and transcription independently proceed to avoid unnecessary collision. A small R-loop will not trigger any specific problem. B) In the presence of excess Spt2p, the transcription rate might be enhanced and multiple RNA polymerases could occupy the transcribed strand to produce a larger R-loop. In addition, Spt2p could bind the junction between the DNA replication fork and the transcription fork. C) Some unknown AID like protein (Gray circle with ?) could introduce uracil by the deamination of cytocine in the large R loop. Uracil would enable the enzyme Ung1p to make a single strand break. Alternatively, the Rad1-Rad10 endonuclease could generate single strand break. D) Chromosomal breaks would lead to GCR formation. RNAP represent RNA polymerase.