On the evolution of thymidine synthesis: lateral gene transfer, viral involvement and early life Adi Stern Under the supervision of Tal Pupko

What makes DNA what it is? Deoxy-nucleotides Deoxy-nucleotides Thymidine Thymidine

Thymidylate Synthase (Thy) ► Synthesizes dTMP (T), one of the DNA building blocks

The canonical Thy enzyme ThyA Almost all Eukarya, E.coli Almost all Eukarya, E.coli The mystery of the slime mold (Mycetozoa) The mystery of the slime mold (Mycetozoa)

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

- ThyX 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 synthases

Goals 1. Quantify and locate events of LGT 2. Find origins of each LGT event using gene trees 3. When did Thy originate?

Studying Thy LGT  Map ThyA and ThyX on the species tree  Reconstruct ThyA / ThyX / both at each ancestor

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 models useful for? X X A A A A A A X A Which one is more likely? X X A A X A A A X A A A A+ X X ThyX gain A ThyA loss ThyX loss A ThyX gain, ThyA loss

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

Likelihood An evolutionary model enables us to compute the likelihood that a certain scenario describes the biological reality we observe. An evolutionary model enables us to compute the likelihood that a certain scenario describes the biological reality we observe. Learn the different probabilities from the data Learn the different probabilities from the data X X A A A A A A X A A+ X X A X X A A X A A A X A A A A

A gain-loss evolutionary model A X A+X AXA+X

An evolutionary model A X A+X AXA+X Rate of gain Rate of loss μ 1, μ 2, μ 3, μ 4 are maximum-likelihood estimates

Results rate of loss >> rate of gain rate of loss >> rate of gain rate of gain of ThyA > gain of ThyX rate of gain of ThyA > gain of ThyX

Results – Maximum Likelihood Reconstruction At least: 14 LGT events where ThyA was swapped by ThyX (or vice versa)

Origin of Mycetozoa ThyX 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 explanations 1. Mycetozoa eat bacteria  digested some bacterial DNA 2. The ancestor of mitochondria is known to be an α-proteobacteria  mitochondrial involvement?

Viruses

Viral sequences ’ distribution Myoviridae Siphoviridiae Herpesviridae Podoviridae

Early life

Beginning of life - RNA world “…The universal ancestor is not a discrete entity. It is, rather, a diverse community of cells that survives and evolves as a biological unit…“ (Woese, 1998) “…The universal ancestor is not a discrete entity. It is, rather, a diverse community of cells that survives and evolves as a biological unit…“ (Woese, 1998) LGT LUCA – the last universal common ancestor, is a communal “swamp”

RNA  DNA world Controversy around how DNA evolved Controversy around how DNA evolved Archaea Bacteria Eukarya

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

Viruses and Thy in early life

ThyA gene tree T4 like phages T4 like phages hosts

ThyA gene tree Bacteria (hosts) Eukarya T4-like phages Archaea ?

A model for Thy evolution in early life

LGT RNA world and RNA viruses

Viruses modify their genome to evade host defense Thymidine Thymidylate synthase

Thymidine dCMP hydroxymethylase )CH( In fact – T4 like phages use a similar tactic today! In fact – T4 like phages use a similar tactic today! Hydroxy- methylated cytosine CH Thymidine

Interestingly, CH and ThyA are homologs (sequentially and structurally) Interestingly, CH and ThyA are homologs (sequentially and structurally) Song et al. EMBO J (18) 1999

Summary Open questions:  When did ThyX come in and how?  Who predated who?  Why is there so much LGT between these two enzymes? ThyA

Thank you …. Dr. Tal Pupko Dr. Tal Pupko Shaul Shaul, Itay Mayrose Shaul Shaul, Itay Mayrose Dr. Uri Gophna Dr. Uri Gophna Lab members: Adi Doron Eyal Privman Nimrod Rubinstein Osnat Penn Dudu Burstein Ofir Cohen Lab members: Adi Doron Eyal Privman Nimrod Rubinstein Osnat Penn Dudu Burstein Ofir Cohen