On the evolution of On the evolution of thymidine synthesis: thymidine synthesis:

lateral gene transfer, viral lateral gene transfer, viral involvement and early lifeinvolvement and early life

Adi SternUnder the supervision of Tal Pupko

What makes DNA what it What makes DNA what it is?is?

Deoxy-nucleotidesDeoxy-nucleotides ThymidineThymidine

Thymidylate Synthase Thymidylate Synthase (Thy)(Thy)

►Synthesizes dTMP (T), one of the DNA building blocksSynthesizes dTMP (T), one of the DNA building blocks

The canonical Thy The canonical Thy enzymeenzyme

ThyA

Almost all Eukarya, E.coliAlmost all Eukarya, E.coli The mystery of the slime mold The mystery of the slime mold (Mycetozoa)(Mycetozoa)

An alternative Thy An alternative Thy enzymeenzyme

In 1989/2002, an alternative mechanism for Thymidylate In 1989/2002, an alternative mechanism for Thymidylate synthesis was discovered, and the enzyme was named synthesis was discovered, and the enzyme was named ThyX ThyX – flavin dependent– flavin dependent

ThyA

ThyX

-

ThyX distributionThyX distribution

Myllykallio et al. Science (297) 2002

Lateral gene

transfer (LGT)

Lateral Gene Transfer (LGT) is any process in which an organism transfers genetic material to another organism that is not its offspring

Evolution of thymidylate Evolution of thymidylate synthasessynthases

GoalsGoals

1.1. Quantify and locate events of LGTQuantify and locate events of LGT

2.2. Find origins of each LGT event using Find origins of each LGT event using gene treesgene trees

3.3. When did Thy originate?When did Thy originate?

Studying Thy LGTStudying Thy LGT

Map ThyA and ThyX on the species treeMap ThyA and ThyX on the species tree Reconstruct Reconstruct ThyAThyA / / ThyXThyX / / bothboth at each at each

ancestorancestor

Legend:- ThyA- ThyX- ThyX & Thy A

Deinococcales: the coloring means there are some deinococci which encode for ThyA and others for ThyX

Thy mapping on species tree:BLAST against ~400 whole genomes

How to reconstruct ?

Evolutionary models

Thy mapping on species tree

What are evolutionary What are evolutionary models useful formodels useful for??

XXA

AA

A

AA

X

A

Which one is more likely?

XXA

A

XA

AA

X

A

AAA+

XX

ThyX gain

A

ThyA loss

ThyX loss

A

ThyX gain, ThyA loss

Evolutionary modelsEvolutionary models

The aim of an evolutionary model: to describe The aim of an evolutionary model: to describe (often in probabilistic terms) the evolutionary (often in probabilistic terms) the evolutionary biological realitybiological reality

87.01.001.002.0

04.068.02.008.0

08.001.08.011.0

08.011.008.073.0

T

G

C

A

TGCA

LikelihoodLikelihood An evolutionary model enables us to An evolutionary model enables us to

compute the compute the likelihoodlikelihood that a certain that a certain scenario describes the biological reality we scenario describes the biological reality we observe.observe.

Learn the different probabilities from the Learn the different probabilities from the datadata

XXA

AA

A

AA

X

A

A+ X

XA

XXA

A

XA

AA

X

A

AA

A

A gain-loss evolutionary A gain-loss evolutionary modelmodel

4333

22

11

0

0

A

X

A+X

A X A+X

An evolutionary modelAn evolutionary model

4333

22

11

0

0

A

X

A+X

A X A+X

Rate of gain

Rate of loss

μ1, μ2, μ3, μ4 are maximum-likelihood estimates

ResultsResults

rate of rate of lossloss >> rate of >> rate of gaingain rate of gain ofrate of gain of ThyAThyA > gain of> gain of ThyXThyX

Results – Maximum Results – Maximum Likelihood ReconstructionLikelihood Reconstruction

At least:

14 LGT events where ThyA was swapped by ThyX (or vice versa)

Origin of Mycetozoa Origin of Mycetozoa ThyXThyX

To find the origin of an LGT, we look at the gene tree based on the multiple sequence alignment of one of the genes

Two possible Two possible explanationsexplanations

1.1. Mycetozoa eat bacteria Mycetozoa eat bacteria digested some bacterial DNAdigested some bacterial DNA

2.2. The ancestor of mitochondria The ancestor of mitochondria is known to be an is known to be an αα-proteobacteria -proteobacteria mitochondrial mitochondrial involvement?involvement?

VirusesViruses

Viral sequences’ distribution

Myoviridae

Siphoviridiae

Herpesviridae

Podoviridae

Early lifeEarly life

Beginning of life - RNA Beginning of life - RNA worldworld

“…“…The universal ancestor is not a discrete The universal ancestor is not a discrete entity. It is, rather, a diverse community of entity. It is, rather, a diverse community of cells that survives and evolves as a cells that survives and evolves as a biological unit…“ (Woese, 1998)biological unit…“ (Woese, 1998)

LGT

LUCA – the last universal common

ancestor, is a communal “swamp”

RNA RNA DNA world DNA world

Controversy around how DNA evolvedControversy around how DNA evolved

Archaea

Bacteria

Eukarya

Founder VirusesFounder Viruses

“…“…Viruses played a critical role in the Viruses played a critical role in the emergence of DNA…several independent emergence of DNA…several independent transfers [from viruses] produced the two cell transfers [from viruses] produced the two cell types (prokaryote/eukaryote) and the three types (prokaryote/eukaryote) and the three cellular domains …” (Forterre PNAS(103) 2006)cellular domains …” (Forterre PNAS(103) 2006)

Archaea

Bacteria

Eukarya

virus

Viruses and Thy in early lifeViruses and Thy in early life

ThyA gene tree

T4 like phages

T4 like phages hosts

ThyA gene treeThyA gene tree

Bacteria(hosts)

Eukarya

T4-like phages

Archaea

?

A model for Thy evolution in A model for Thy evolution in early lifeearly life

LGT

RNA world and RNA RNA world and RNA virusesviruses

Viruses modify their Viruses modify their genome to evade host genome to evade host

defensedefense

Thymidine

Thymidylate synthase

Thymidine

dCMP dCMP hydroxymethylase hydroxymethylase ))CHCH((

In fact – T4 like phages use a similar tactic In fact – T4 like phages use a similar tactic today!today!

Hydroxy-methylated cytosine

CH

Thymidine

Interestingly, CH and ThyA are homologs Interestingly, CH and ThyA are homologs (sequentially and structurally)(sequentially and structurally)

Song et al. EMBO J (18) 1999

SummarySummary

Open questions:

When did ThyX come in and how?

Who predated who?

Why is there so much LGT between these two enzymes?ThyA

Thank youThank you.… .… Dr. Tal PupkoDr. Tal Pupko Shaul Shaul, Itay MayroseShaul Shaul, Itay Mayrose Dr. Uri GophnaDr. Uri Gophna

Lab members:Lab members:Adi DoronAdi DoronEyal PrivmanEyal PrivmanNimrod RubinsteinNimrod RubinsteinOsnat PennOsnat PennDudu BursteinDudu BursteinOfir CohenOfir Cohen