How do Replication and Transcription Change Genomes? Andrey Grigoriev Director, Center for Computational and Integrative Biology Rutgers University

2 What are we going to do? Observe effects of fundamental processes Estimate their relative contribution Link them to genome features Analyze nucleotide composition

How do Replication and Transcription Change Genomes? Well, do they?

4 Replication and Transcription textbook view faithful reproduction machinery basis for selection parental DNA  fitness advantages

5 Replication and Transcription paradox both systematically change genomes which they faithfully reproduce and they leave traces

6 What is in the sequence? The usual –coding, regulatory regions, exons, introns, RNAs, etc. Biases in nucleotide composition –Traces of organism‘s „lifestyle“ –Links to genome features

7 Counting nucleotides: GC Skew s w = ([G]-[C])/([G]+[C]) Short sequence interval (window) w Relative excess of G vs C [-1;1] Plot vs % of genome position [0;100]

8 position, % genome length Simian virus 40 Haemophilis influenzae

9 Cumulative Skew Diagrams s w = ([G]-[C])/([G]+[C]) S =  W s w w/L For W adjacent windows of size w << L S is an integral of skew function

10 Simian virus 40 replication origin (ori) replication terminus (ter)

11 Haemophilis influenzae replication origin (ori) replication terminus (ter)

12 Genome of Escherichia coli position, % genome length Terminus Origin

13 Genome of Bacillus subtilis

14 Genome of Borellia burgdorferi position, % genome length

15 Cumulative Skew Diagrams Now widely used to predict ori and ter in novel and less studied microbial genomes Predictions confirmed experimentally Constant skews over half-genomes ori  ter G>C ter  ori G<C Strand properties change at ori and ter

16 Causes: Selection vs. Mutation Properties of encoded proteins Regulatory sequences Most pronounced in 3 rd codon position Suggests mutation, not selection pressure

17 DNA single-stranded, not protected continuous DNA synthesis discontinuous DNA synthesismRNA synthesis template DNA Transcription Replication

18 Most Consistent Explanation spontaneous deamination of C or 5-MetC –by far the most frequent mutation (rates raise over 100-fold when DNA is single-stranded) –fixing the mutated base during the next round of replication –depletion of cytosines vs guanines

19 Cytosine Deamination Cytosine Uracil Thymine

20 Replication Leading strand exposed in replication bubble, generation after generation Unusual replication models consistent with the single-strand hypothesis –adenovirus –mitochondria

21 position, % genome length Adenovirus Replication origins

22 Replication or Transcription Leading-lagging switch at ori and ter Consistent with replication models Transcription often colinear with replication Direction often changes at ori and ter

23 Replication vs. Transcription HPV-16

24 Replication vs. Transcription Comparable contribution to skew [G]=900, [C]=690 in the same direction additive effect on skew [G]=758, [C]=773 in the opposite direction cancel each other out

25 Genome of Bacillus subtilis

26 Diagrams „jagged“ Sequence constraints –amino acid composition, regulatory sequences, etc. Sequence inversions –swaps strands and change the skew to its opposite between the borders of the inversion Horizontal transfer between species

27 5‘3‘ A B C DA C B D 3‘5‘ Inversion

28 Rearrangements in two sequenced strains of Helicobacter pylori Colored areas under the curve correspond to inversions and translocations cagPAI – pathogenicity island (likely horizontal transfer)

29 Conclusions Analyze nucleotide composition Observe effects of fundamental processes Link them to genome features Estimate their relative contribution Start asking own questions