How do Replication and
Transcription Change Genomes?
Andrey Grigoriev
Director, Center for Computational and Integrative Biology
Rutgers University
What are we going to do?
• Observe effects of fundamental processes
• Estimate their relative contribution
• Link them to genome features
• Analyze nucleotide composition
2
How do Replication and
Transcription Change Genomes?
Well, do they?
Replication and Transcription
• textbook view
faithful reproduction machinery
• basis for selection
parental DNA  fitness advantages
4
Replication and Transcription
• paradox
both systematically change genomes
which they faithfully reproduce
• and they leave traces
5
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
6
Counting nucleotides: GC Skew
sw = ([G]-[C])/([G]+[C])
• Short sequence interval (window) w
• Relative excess of G vs C
[-1;1]
• Plot vs % of genome position [0;100]
7
Simian virus 40
0
20
40
60
80
100
0
20
40
60
80
100
Haemophilis influenzae
position, % genome length
8
Cumulative Skew Diagrams
sw = ([G]-[C])/([G]+[C])
S = W sw w/L
For W adjacent windows of size w << L
S is an integral of skew function
9
Simian virus 40
0
20
40
60
0
20
40
60
80
100
replication origin (ori)
replication terminus (ter)
80
position, % genome length
10
100
Haemophilis influenzae
0
20
40
60
80
100
0
20
40
60
80
100
replication origin (ori)
replication terminus (ter)
position, % genome length
11
Genome of Escherichia coli
Terminus
0
20
40
60
80
Origin
position, % genome length
12
100
Genome of Bacillus subtilis
0
20
60
40
position, % genome length
13
80
100
Genome of Borellia burgdorferi
0
20
40
60
position, % genome length
14
80
100
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
15
Causes: Selection vs. Mutation
• Properties of encoded proteins
• Regulatory sequences
• Most pronounced in 3rd codon position
• Suggests mutation, not selection pressure
16
Transcription
Replication
template DNA
mRNA synthesis
continuous DNA synthesis
discontinuous DNA synthesis
DNA single-stranded, not protected
17
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
18
Cytosine Deamination
Uracil
Cytosine
Thymine
19
Replication
• Leading strand exposed in replication
bubble, generation after generation
• Unusual replication models consistent with
the single-strand hypothesis
– adenovirus
– mitochondria
20
Adenovirus Replication
origins
0
20
40
60
80
position, % genome length
21
100
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
22
Replication vs. Transcription
HPV-16
0
20
40
60
position, % genome length
23
80
100
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
24
Genome of Bacillus subtilis
0
20
60
40
position, % genome length
25
80
100
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
26
Inversion
5‘
A B
3‘
C
5‘
D
A C
27
3‘
B
D
Rearrangements in two
sequenced strains of
Helicobacter pylori
Colored areas under the
curve correspond to
inversions and
translocations
cagPAI – pathogenicity
island (likely horizontal
transfer)
28
Conclusions
•
•
•
•
Analyze nucleotide composition
Observe effects of fundamental processes
Link them to genome features
Estimate their relative contribution
• Start asking own questions
29