Recombinogenicity of palindromes and quasipalindromes in the yeast Saccharomyces cerevisiae genome

U ovom radu istražena je rekombinogenost savršenih palindroma i kvazipalindroma s kratkim razmaknicama u genomu kvasca Saccharomyces cerevisiae. Pokazano je da minimalna duljina razmaknice koja statistički značajno smanjuje rekombinogenost palindroma duljine 126 pb iznosi 6 pb, a razmaknica duljine 10 pb u potpunosti anulira rekombinogenost palindroma duljine do 150 pb. Nadalje, pokazano je da na rekombinogenost palindroma in vivo može utjecati temperatura okoline. Osim toga, rekombinogenost palindroma i kvazipalindroma istražena je u nizu mutanata kvasca, te je utvrđeno da helikaza Sgs1 smanjuje rekombinogenost kvazipalindroma i savršenih palindroma, dok endonukleaza Rad27 smanjuje rekombinogenost i učestalost gubitka savršenih palindroma. Nadalje, pokazno je da protein Ku70 povećava rekombinogenost palindroma in vivo, najvjerojatnije stabilizacijom strukture kruciforma. Također, konstruiran je eksperimentalni sustav u kojem rekombinogenost savršenih palindroma korelira s intenzitetom boje kolonija kvasca, a može se koristiti i za identifikaciju novih gena/proteina uključenih u rekombinogenost palindroma na temelju komplementacije mutacije u mutantima dobivenim nasumičnom mutagenezom.In this thesis recombinogenicity of perfect palindromes and quasipalindromes in the yeast Saccharomyces cerevisiae genome was investigated. It was shown that a minimal spacer length which influences recombinogenicity of a 126 bp palindrome is 6 bp, while 10 bp long spacer completely abolishes recombinogenicity of palindromes up to 150 bp long. Furthermore, it was shown that growth temperature can affect palindrome recombinogenicity in vivo. Palindrome recombinogenicity was investigated in a series of yeast mutants and it was shown that helicase Sgs1 reduces recombinogenicity of both perfect palindromes and quasipalindromes, while endonuclease Rad27 reduces recombinogenicity and deletion frequency of perfect palindromes. On the other hand, protein Ku70 increases palindrome recombinogenicity, possibly by stabilisation of cruciform structures. Furthermore, an experimental system based on correlation between yeast colony colour and palindrome recombinogenicity was developed and it was shown that this system can be used to identify novel proteins responsible for palindrome recombinogenicity using complementation of mutants obtained by random mutagenesis