Where in a Genome Does DNA Replication

Begin?
Algorithmic Warm-Up

Phillip Compeau and Pavel Pevzner

Bioinformatics Algorithms: an Active Learning Approach
©2013 by Compeau and Pevzner. All rights reserved
Before a Cell Divides, it Must Replicate its
Genome
Replication begins in a region called
the replication origin (oriC)

Where in a genome does it all

begin?
 Outline
• Search for Hidden Messages in Replication Origin
– What is a Hidden Message in Replication Origin?
– Some Hidden Messages are More Surprising than Others
– Clumps of Hidden Messages
• From a Biological Insight toward an Algorithm for Finding
Replication Origin
– Asymmetry of Replication
– Why would a computer scientist care about assymetry of
replication?
– Skew Diagrams
– Finding Frequent Words with Mismatches
– Open Problems
 Finding Origin of Replication

Finding oriC Problem: Finding oriC in a genome.

• Input. A genome.
• Output. The location of oriC in the genome.

OK – let’s cut out this DNA This is not a

fragment. Can the genome replicate computational
without it? problem!
 How Does the Cell Know to Begin
Replication in Short oriC?
Replication origin of Vibrio cholerae (≈500
nucleotides):
atcaatgatcaacgtaagcttctaagcatgatcaaggtgctcacacagtttatccacaac
ctgagtggatgacatcaagataggtcgttgtatctccttcctctcgtactctcatgacca
cggaaagatgatcaagagaggatgatttcttggccatatcgcaatgaatacttgtgactt
gtgcttccaattgacatcttcagcgccatattgcgctggccaaggtgacggagcgggatt
acgaaagcatgatcatggctgttgttctgtttatcttgttttgactgagacttgttagga
tagacggtttttcatcactgactagccaaagccttactctgcctgacatcgaccgtaaat
tgataatgaatttacatgcttccgcgacgatttacctcttgatcatcgatccgattgaag
atcttcaattgttaattctcttgcctcgactcatagccatgatgagctcttgatcatgtt
tccttaaccctctattttttacggaagaatgatcaagctgctgctcttgatcatcgtttc

There must be a hidden message telling the cell to start

replication here.
 The Hidden Message Problem
Hidden Message Problem. Finding a hidden
message in a string.
• Input. A string Text (representing replication
origin).
• Output. A hidden message in Text.
This is not a
computational
problem either!
The notion of “hidden message” is not
precisely defined.
 The Hidden Message Problem
Revisited
Hidden Message Problem. Finding a hidden
message in a string.
• Input. A string Text (representing oriC).
• Output. A hidden message in Text.

The notion of “hidden message” is

This is not a
computational not precisely defined.
problem either!
Hint: For various biological signals, certain
words appear surprisingly frequently in
small regions of the genome.

AATTT is a surprisingly frequent 5-mer in:

 The Frequent Words Problem
Frequent Words Problem. Finding most frequent k-mers in a
string.
• Input. A string Text and an integer k.
• Output. All most frequent k-mers in Text.
This is better, but where
is the definition of “a
most frequent k-mer?”
 The Frequent Words Problem
Frequent Words Problem. Finding most frequent k-mers in a
string.
• Input. A string Text and an integer k.
• Output. All most frequent k-mers in Text.
A k-mer Pattern is a most frequent k-mer
in a text if no other k-mer is more frequent
than Pattern.

AATTT is a most frequent 5-mer in:

ACAAATTTGCATAATTTCGGGAAATTTCCT

Son Pham, Ph.D., kindly gave us

permission to use his photographs
and greatly helped with preparing
this presentation. Thank you Son!
 Does the Frequent Words Problem
Make Sense to Biologists?
Frequent Words Problem. Finding most frequent k-mers in a
string.
• Input. A string Text and an integer k.
• Output. All most frequent k-mers in Text.
Replication is performed by DNA polymerase and the initiation
of replication is mediated by a protein called DnaA.

DnaA binds to short (typically 9 nucleotides long) segments

within the replication origin known as a DnaA box.

A DnaA box is a hidden message telling DnaA: “bind here!”

And DnaA wants to see multiple DnaA boxes.
 Outline
• Search for Hidden Messages in Replication Origin
– What is a Hidden Message in Replication Origin?
– Some Hidden Messages are More Surprising than Others
– Clumps of Hidden Messages
• From a Biological Insight toward an Algorithm for Finding
Replication Origin
– Asymmetry of Replication
– Why would a computer scientist care about assymetry of
replication?
– Skew Diagrams
– Finding Frequent Words with Mismatches
– Open Problems
 oriC of Vibrio cholerae

atcaatgatcaacgtaagcttctaagcatgatcaaggtgctcacacagtttatccacaacctgagtgg
atgacatcaagataggtcgttgtatctccttcctctcgtactctcatgaccacggaaagatgatcaag
agaggatgatttcttggccatatcgcaatgaatacttgtgacttgtgcttccaattgacatcttcagc
gccatattgcgctggccaaggtgacggagcgggattacgaaagcatgatcatggctgttgttctgttt
atcttgttttgactgagacttgttaggatagacggtttttcatcactgactagccaaagccttactct
gcctgacatcgaccgtaaattgataatgaatttacatgcttccgcgacgatttacctcttgatcatcg
atccgattgaagatcttcaattgttaattctcttgcctcgactcatagccatgatgagctcttgatca
tgtttccttaaccctctattttttacggaagaatgatcaagctgctgctcttgatcatcgtttc
 Too Many Frequent Words – Which
One is a Hidden Message?

atcaatgatcaacgtaagcttctaagcATGATCAAGgtgctcacacagtttatccacaacctgagtgg
atgacatcaagataggtcgttgtatctccttcctctcgtactctcatgaccacggaaagATGATCAAG
agaggatgatttcttggccatatcgcaatgaatacttgtgacttgtgcttccaattgacatcttcagc
gccatattgcgctggccaaggtgacggagcgggattacgaaagcatgatcatggctgttgttctgttt
atcttgttttgactgagacttgttaggatagacggtttttcatcactgactagccaaagccttactct
gcctgacatcgaccgtaaattgataatgaatttacatgcttccgcgacgatttacctCTTGATCATcg
atccgattgaagatcttcaattgttaattctcttgcctcgactcatagccatgatgagctCTTGATCA
TgtttccttaaccctctattttttacggaagaATGATCAAGctgctgctCTTGATCATcgtttc

Most frequent 9-mers in this oriC (all appear 3 times):

ATGATCAAG, CTTGATCAT, TCTTGGATCA, CTCTTGATC

Is it STATISTICALLY surprising to find a 9-mer appearing 3

or more times within ≈ 500 nucleotides?
 Hidden Message Found!

atcaatgatcaacgtaagcttctaagcATGATCAAGgtgctcacacagtttatccacaacctgagtgg
atgacatcaagataggtcgttgtatctccttcctctcgtactctcatgaccacggaaagATGATCAAG
agaggatgatttcttggccatatcgcaatgaatacttgtgacttgtgcttccaattgacatcttcagc
gccatattgcgctggccaaggtgacggagcgggattacgaaagcatgatcatggctgttgttctgttt
atcttgttttgactgagacttgttaggatagacggtttttcatcactgactagccaaagccttactct
gcctgacatcgaccgtaaattgataatgaatttacatgcttccgcgacgatttacctCTTGATCATcg
atccgattgaagatcttcaattgttaattctcttgcctcgactcatagccatgatgagctCTTGATCA
TgtttccttaaccctctattttttacggaagaATGATCAAGctgctgctCTTGATCATcgtttc

ATGATCAAG
||||||||| are reverse complements and likely DnaA
boxes
TACTAGTTC (DnaA does not care what strand to bind to)

It is VERY SURPRISING to find a 9-mer appearing 6 or more

times (counting reverse complements) within a short ≈ 500
 Can we Now Find Hidden Messages in
Thermotoga petrophila?
aactctatacctcctttttgtcgaatttgtgtgatttatagagaaaatcttattaactgaaactaa
aatggtaggtttggtggtaggttttgtgtacattttgtagtatctgatttttaattacataccgta
tattgtattaaattgacgaacaattgcatggaattgaatatatgcaaaacaaacctaccaccaaac
tctgtattgaccattttaggacaacttcagggtggtaggtttctgaagctctcatcaatagactat
tttagtctttacaaacaatattaccgttcagattcaagattctacaacgctgttttaatgggcgtt
gcagaaaacttaccacctaaaatccagtatccaagccgatttcagagaaacctaccacttacctac
cacttacctaccacccgggtggtaagttgcagacattattaaaaacctcatcagaagcttgttcaa
aaatttcaatactcgaaacctaccacctgcgtcccctattatttactactactaataatagcagta
taattgatctgaaaagaggtggtaaaaaa

No single occurrence of ATGATCAAG or CTTGATCAT from

Vibrio Cholerae!!!
Applying the Frequent Words Problem to this replication
origin:
AACCTACCA, ACCTACCAC, GGTAGGTTT, TGGTAGGTT,
AAACCTACC, CCTACCACC
Different genomes → different hidden messages (DnaA
 Hidden Messages in Thermotoga
petrophila
aactctatacctcctttttgtcgaatttgtgtgatttatagagaaaatcttattaactgaaactaa
aatggtaggtttGGTGGTAGGttttgtgtacattttgtagtatctgatttttaattacataccgta
tattgtattaaattgacgaacaattgcatggaattgaatatatgcaaaacaaaCCTACCACCaaac
tctgtattgaccattttaggacaacttcagGGTGGTAGGtttctgaagctctcatcaatagactat
tttagtctttacaaacaatattaccgttcagattcaagattctacaacgctgttttaatgggcgtt
gcagaaaacttaccacctaaaatccagtatccaagccgatttcagagaaacctaccacttacctac
cacttaCCTACCACCcgggtggtaagttgcagacattattaaaaacctcatcagaagcttgttcaa
aaatttcaatactcgaaaCCTACCACCtgcgtcccctattatttactactactaataatagcagta
taattgatctgaaaagaggtggtaaaaaa

Ori-Finder software confirms that

CCTACCACC
||||||||| are candidate hidden messages.
GGATGGTGG

We learned how to find hidden messages IF oriC is given. But

we have no clue WHERE oriC is located in a (long) genome.
 Outline
• Search for Hidden Messages in Replication Origin
– What is a Hidden Message in Replication Origin?
– Some Hidden Messages are More Surprising than Others
– Clumps of Hidden Messages
• From a Biological Insight toward an Algorithm for Finding
Replication Origin
– Asymmetry of Replication
– Why would a computer scientist care about assymetry of
replication?
– Skew Diagrams
– Finding Frequent Words with Mismatches
– Open Problems
 Finding Replication Origin

Our strategy BEFORE: given a previously known oriC (a

500-nucleotide window), find frequent words (clumps)
in oriC as candidate DnaA boxes.
replication origin → frequent words
 Finding Replication Origin

Our strategy BEFORE: given previously known oriC (a

500-nucleotide window), find frequent words (clumps)
in oriC as candidate DnaA boxes.
replication origin → frequent words

But what if the position of the replication origin within a

genome is unknown!
 Finding Replication Origin

Our strategy BEFORE: given previously known oriC (a

500-nucleotide window), find frequent words (clumps)
in oriC as candidate DnaA boxes.
replication origin → frequent words

NEW strategy: find frequent words in ALL windows within a

genome. Windows with clumps of frequent words are
candidate replication origins.
frequent words → replication origin
 What is a Clump?
Formal: AAk-mer
Intuitive: k-merforms
formsan
a clump
(L, t)-clump
insideinside
Genome
Genome
if there
if
is a short
there is a short
interval
(length
of Genome
L) interval
in which
of Genome
it appears
in which
manyit
times. many (at least t) times.
appears

Clump Finding Problem. Find patterns forming clumps in

a string.
• Input. A string Genome and integers k (length of a
pattern), L (window length), and t (number of patterns in
a clump).
• Output. All k-mers forming (L, t)-clumps in Genome.
There exist 1904 different 9-mers forming (500,3)-clumps in
E. coli genome. It is absolutely unclear which of them
point to the replication origin…
 Where in a Genome Does DNA Replication
Begin?
Algorithmic Warm-Up

Phillip Compeau and Pavel Pevzner

Bioinformatics Algorithms: an Active Learning Approach
©2013 by Compeau and Pevzner. All rights reserved
 Outline
• Search for Hidden Messages in Replication Origin
– What is a Hidden Message in Replication Origin?
– Some Hidden Messages are More Surprising than Others
– Clumps of Hidden Messages
• From a Biological Insight toward an Algorithm for Finding
Replication Origin
– Asymmetry of Replication
– Why would a computer scientist care about assymetry of
replication?
– Skew Diagrams
– Finding Frequent Words with Mismatches
– Open Problems
DNA Strands Have Directions!

5’ oriC 3’

3’ oriC 5’

The two strands run in opposite

directions
(from 5’ to 3’):
Blue Strand Clockwise,
Green Strand Counter-Clockwise
terC

terC
DNA Strands Have Directions

5’ oriC 3’

3’ oriC 5’

If you were a DNA Polymerase,

how would you replicate a
genome???

terC

terC
Four DNA Polymerases Do the Job
oriC

5’ 3’

3’ 5’

oriC

terC

terC
Continue as Replication Fork
Enlarges
5’ 3’

3’ 5’

Simple but wrong: DNA polymerases

are unidirectional: they can only
traverse a parent strand in the opposite
(3’ → 5’) direction.
 If you Were a UNIDIRECTIONAL DNA
Polymerase, how Would you Replicate a
Genome?

5’ 3’

3’ 5’

Reverse Forward Reverse Forward

half-strand half-strand half- half-strand
strand

Big problemreplicating
No problem replicatingreverse
forwardhalf-strands
half-strands (thin
 If you Were a UNIDIRECTIONAL DNA
Polymerase, How Would you Replicate a
Genome???
5’ 3’

3’ 5’

Reverse Forward Reverse Forward

half-strand half-strand half- half-strand
strand
Wait until the Fork Opens and…

5’ 3’

3’ 5’
Wait until the Fork Opens and
Replicate
5’ 3’

3’ 5’
 Replicate
Wait until the Fork Opens Even More
and…

Okazaki
fragments
 Replicate
Wait until the Fork Opens Even More
and… Okazaki
fragments

Okazaki
fragments

REPLICA
TE!

Instead of copying the entire half-strand, many Okazaki fragments are

Okazaki Fragments Need to be Ligated to
Fill in the Gaps

Okazaki
fragments

The genome has been replicated!

 Different Lifestyles of Reverse and
Forward Half-Strands
The reverse half-strand lives a
waitin
double-stranded life most of g
the time.
waitin
The forward half-strand g
spends a large portion of its life
single-stranded, waiting to be
replicated.

But why would a computer scientist

care?
 Outline
• Search for Hidden Messages in Replication Origin
– What is a Hidden Message in Replication Origin?
– Some Hidden Messages are More Surprising than Others
– Clumps of Hidden Messages
• From a Biological Insight toward an Algorithm for Finding
Replication Origin
– Asymmetry of Replication
– Why would a computer scientist care about assymetry of
replication?
– Skew Diagrams
– Finding Frequent Words with Mismatches
– Open Problems
 Asymmetry of Replication Affects
Nucleotide Frequencies

Single-stranded DNA has a much higher mutation rate

than double-stranded DNA.

Thus, if one nucleotide has a greater mutation rate, then

we should observe its shortage on the forward half-strand
that lives single-stranded life!

Which nucleotide (A/C/G/T) has the highest mutation rate?

Why?
 The Peculiar Statistics of #G - #C
Cytosine (C) rapidly mutates into thymine (T) through
deamination; deamination rates rise 100-fold when DNA is
single stranded!
Forward half-strand (single-stranded life): shortage of C,
normal G
Reverse half-strand (double-stranded life): shortage of G,
normal C
#C #G
#G - #C
Reverse half-strand 219518 201634 -
17884
Forward half-strand 207901 211607
+3706
Difference +11617 -9973
 Take a Walk Along the Genome
#G - #C is #G - #C is
DECREASING 5’ 3’
INCREASING
3’ oriC 5’

C high C low
G low You walk along the genome and see that #G - #C G high
have been decreasing and then suddenly starts
increasing.

WHERE ARE YOU IN THE

GENOME? terC

C high/G low → #G - #C is C low/G high → #G - #C is INCREASING

DECREASING as we walk along the as we walk along the FORWARD half-
REVERSE half-strand strand
 Outline
• Search for Hidden Messages in Replication Origin
– What is a Hidden Message in Replication Origin?
– Some Hidden Messages are More Surprising than Others
– Clumps of Hidden Messages
• From a Biological Insight toward an Algorithm for Finding
Replication Origin
– Asymmetry of Replication
– Why would a computer scientist care about assymetry of
replication?
– Skew Diagrams
– Finding Frequent Words with Mismatches
– Open Problems
 Skew Diagram

Skew(k): #G - #C for the first k nucleotides of

Genome.

Skew diagram: Plot Skew(k) against k

CATGGGCATCGGCCATACGCC
 Skew Diagram of E. Coli:
Where is the Origin of Replication?

ori
C

You walk along the genome and see that #G - #C have been decreasing
and then suddenly starts increasing: WHERE ARE YOU IN THE
We Found the Replication Origin in E. Coli
BUT…
The minimum of the Skew Diagram
points to this region in E. coli:
aatgatgatgacgtcaaaaggatccggataaaacatggtgattgcctcgcataacgcggta
tgaaaatggattgaagcccgggccgtggattctactcaactttgtcggcttgagaaagacc
tgggatcctgggtattaaaaagaagatctatttatttagagatctgttctattgtgatctc
ttattaggatcgcactgccctgtggataacaaggatccggcttttaagatcaacaacctgg
aaaggatcattaactgtgaatgatcggtgatcctggaccgtataagctgggatcagaatga
ggggttatacacaactcaaaaactgaacaacagttgttctttggataactaccggttgatc
caagcttcctgacagagttatccacagtagatcgcacgatctgtatacttatttgagtaaa
ttaacccacgatcccagccattcttctgccggatcttccggaatgtcgtgatcaagaatgt
tgatcttcagtg

But there are no frequent 9-mers (that appear

three or more times) in this region!
SHOULD WE GIVE UP?
 Outline
• Search for Hidden Messages in Replication Origin
– What is a Hidden Message in Replication Origin?
– Some Hidden Messages are More Surprising than Others
– Clumps of Hidden Messages
• From a Biological Insight toward an Algorithm for Finding
Replication Origin
– Asymmetry of Replication
– Why would a computer scientist care about assymetry of
replication?
– Skew Diagrams
– Finding Frequent Words with Mismatches
– Open Problems
 Searching for Even More Elusive
Hidden Messages

atcaatgatcaacgtaagcttctaagcATGATCAAGgtgctcacacagtttatccacaac
ctgagtggatgacatcaagataggtcgttgtatctccttcctctcgtactctcatgacca
cggaaagATGATCAAGagaggatgatttcttggccatatcgcaatgaatacttgtgactt
gtgcttccaattgacatcttcagcgccatattgcgctggccaaggtgacggagcgggatt
acgaaagcatgatcatggctgttgttctgtttatcttgttttgactgagacttgttagga
tagacggtttttcatcactgactagccaaagccttactctgcctgacatcgaccgtaaat
tgataatgaatttacatgcttccgcgacgatttacctCTTGATCATcgatccgattgaag
atcttcaattgttaattctcttgcctcgactcatagccatgatgagctCTTGATCATgtt
tccttaaccctctattttttacggaagaATGATCAAGctgctgctCTTGATCATcgtttc

oriC in Vibrio cholerae has 6 DnaA boxes – can you find

more?
Previously Invisible DnaA Boxes
oriC in Vibrio cholerae contains ATGATCAAC and
CATGATCAT, which differ from canonical DnaA
boxes ATGATCAAG/CTTGATCAT in a single
mutation:
atcaATGATCAACgtaagcttctaagcATGATCAAGgtgctcacacagtttatccacaac
ctgagtggatgacatcaagataggtcgttgtatctccttcctctcgtactctcatgacca
cggaaagATGATCAAGagaggatgatttcttggccatatcgcaatgaatacttgtgactt
gtgcttccaattgacatcttcagcgccatattgcgctggccaaggtgacggagcgggatt
acgaaagCATGATCATggctgttgttctgtttatcttgttttgactgagacttgttagga
tagacggtttttcatcactgactagccaaagccttactctgcctgacatcgaccgtaaat
tgataatgaatttacatgcttccgcgacgatttacctCTTGATCATcgatccgattgaag
atcttcaattgttaattctcttgcctcgactcatagccatgatgagctCTTGATCATgtt
tccttaaccctctattttttacggaagaATGATCAAGctgctgctCTTGATCATcgtttc

Frequent Words with Mismatches Problem. Find the most

frequent k-mers with mismatches in a string.
• Input. A string Text, and integers k and d.
• Output. All most frequent k-mers with up to d mismatches in
Text.
 Finally, DnaA Boxes in E. Coli!
Frequent 9-mers (with 1 Mismatch and
Reverse Complements) in putative oriC of E.
coli
aatgatgatgacgtcaaaaggatccggataaaacatggtgattgcctcgcataacgcggt
atgaaaatggattgaagcccgggccgtggattctactcaactttgtcggcttgagaaaga
cctgggatcctgggtattaaaaagaagatctatttatttagagatctgttctattgtgat
ctcttattaggatcgcactgcccTGTGGATAAcaaggatccggcttttaagatcaacaac
ctggaaaggatcattaactgtgaatgatcggtgatcctggaccgtataagctgggatcag
aatgaggggTTATACACAactcaaaaactgaacaacagttgttcTTTGGATAACtaccgg
ttgatccaagcttcctgacagagTTATCCACAgtagatcgcacgatctgtatacttattt
gagtaaattaacccacgatcccagccattcttctgccggatcttccggaatgtcgtgatc
aagaatgttgatcttcagtg
 Complications
• Some bacteria have fewer DnaA boxes.
• Terminus of replication is often not located
directly opposite to oriC.
• The skew diagram is often more complex
than in the case of E. coli.

The skew diagram of Thermotoga

 Outline
• Search for Hidden Messages in Replication Origin
– What is a Hidden Message in Replication Origin?
– Some Hidden Messages are More Surprising than Others
– Clumps of Hidden Messages
• From a Biological Insight toward an Algorithm for Finding
Replication Origin
– Asymmetry of Replication
– Why would a computer scientist care about assymetry of
replication?
– Skew Diagrams
– Finding Frequent Words with Mismatches
– Open Problems: From Massive Open Online Courses
(MOOC) to Massive Open Online Research (MOOR)
 Finding Multiple Origins of Replication in a
Bacterial Genome
• Biologists long believed that each bacterial chromosome
has a single replication origin.
• Xia (2012) argued that some bacteria may have multiple
replication origins.
Open Problem: Can you
confirm or refute the Xia
conjecture that this bacterial
oriC? oriC? genome indeed has multiple
replication origins?
Project
Director
Mikhail
Skew diagram of Wigglesworthia Gelfand
glossinidia
 Finding oriC in Archaea
oriC

oriC

oriC

Project
Director
Mikhail
The skew diagram for Sulfolocus salfataricus Gelfand

Open Problem: Archaea do have multiple origins of

replication (3 in Sulfolocus salfataricus) but there is no
algorithm and software tool yet to predict them reliably –
 Finding oriC in Yeast

If you feel that finding bacterial replication origins is difficult,

wait until you analyze replication origins in yeast or
humans.

Open Problem: Yeast genomes have

hundreds of origins of replication, but
there is no software tool to predict them
reliably – can you develop such a tool?

Project
Director
Uri Keich
Happy Rosalind!