Nucleosome Positioning Determinants

Histone posttranslational modifications have been associated with changes in chromatin structure necessary for transcription, replication, and DNA repair. Acetylation is one of the most studied and best characterized histone posttranslational modifications, but it is not known if histone acetylation modulates base excision repair of DNA lesions in chromatin. To address this question, we generated nucleosome core particles (NCPs) containingsite-specificallyacetylatedH3K14orH3K56andmea-sured repair of uracil and single-nucleotide gaps. We find that H3K56Ac and H3K14Ac do not significantly contribute to removal of uracils by uracil DNA glycosylase regardless of the translational or rotational position of the lesions within NCPs. In repair of single-nucleotide gaps, however, the presence of H3K56Ac or H3K14Ac in NCPs decreases the gap-filling activity of DNA polymerase ␤ near the dyad center, with H3K14Ac exhibiting stronger inhibition. To a lesser extent, H3K56Ac induces a similar effect near the DNA ends. Moreover, using restriction enzyme accessibility, we detect no changes in NCP structure or dynamics between H3K14Ac-NCPs and WT-NCPs containing single-nucleotide gaps. Thus, acetylation at H3K56 and H3K14 in nucleosomes may promote alternative gap-filling pathways by inhibiting DNA polymerase ␤ activity.Eukaryotic DNA is organized into arrays of nucleosomes, which constitute the primary level of chromatin compaction. Vital DNA-templated processes, including transcription and DNA repair, are dependent on protein-DNA interactions, which are restricted by the histone proteins in nucleosomes. The nucleosome core particle (NCP) 3 consists of 147 bp of DNA wrapped ϳ1.7 times around a histone octamer composed of a tetramer of histones H3 and H4 ((H3-H4) 2 ) flanked by two heterodimers of histones H2A and H2B (H2A-H2B) (1). Accessibility of nuclear factors to occluded DNA in this stable, yet dynamic protein-DNA complex is regulated by multiple mechanisms, including the activity of chromatin remodeling complexes (2, 3) and the intrinsic, partial unwrapping of DNA ends from the histone octamer (4, 5).The rate of DNA unwrapping, also referred to as DNA breathing, in nucleosomes is dependent on DNA sequence and the posttranslational modification (PTM) status of the histones (6 -8

Section: Methodsmentioning

confidence: 99%

Site-specific Acetylation of Histone H3 Decreases Polymerase β Activity on Nucleosome Core Particles in Vitro

Rodriguez¹,

Hinz

Laughery

et al. 2016

“…A total of seven oligonucleotides were purchased, six of which contained a single uracil in the strand corresponding to the I chain in the NCP 601 crystal structure reported by Vasudevan et al (42). Nomenclature of the substrates follows the same convention as reported by Fernandez and Anderson (41), where "ϩ" and "Ϫ" indicate the number of nucleotides away from the dyad toward the 5Ј and 3Ј ends, respectively. Each of these oligonucleotides was radiolabeled with [␥- 32 P]ATP (PerkinElmer Life Sciences) at the 5Ј end using T4 polynucleotide kinase (Invitrogen) and was annealed (1:1) with its complementary or partner strand by heating to 95°C for 10 min and slow cooling in a buffer containing 30 mM Tris (pH 7.5) and 100 mM potassium acetate.…”

Section: Methodsmentioning

confidence: 99%

“…Although TA dinucleotides in NCPs tend to compress the minor groove width (42), thus predicting a disadvantage for enzymes like UDG and pol ␤ that make minor groove contacts with DNA (11,37), it has nevertheless been observed that the activity of UDG is enhanced by the presence of local DNA flexibility (62). Additionally, proteins that bind to the minor groove have less information available and presumably utilize indirect modes that are dependent on the intrinsic shape and mechanical properties of DNA (41). It is conceivable that this structural feature might be responsible for increased recognition by the glycosylase.…”

Section: A Andmentioning

confidence: 99%

The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes

Rodriguez

Smerdon

2013

126

Background: Base excision repair is hindered by nucleosomes. Results: Outwardly oriented uracils near the nucleosome center are efficiently cleaved; however, polymerase ␤ is strongly inhibited at these sites. Conclusion:The histone octamer presents different levels of constraints on BER, dependent on the structural requirements for enzyme activity. Significance: Chromatin remodeling is necessary to prevent accumulation of aborted intermediates in nucleosomes.

“…Mononucleosomes (NCPs) were prepared by histone octamer transfer as described previously (18,21). Three pmol of radiolabeled 147-bp DNA substrates were combined with 300 pmol of chicken erythrocyte core particles prepared from chicken erythrocytes (29) at high ionic strength, and reconstituted by subsequent incremental dialysis (30) (Fig. 1B).…”

Section: Methodsmentioning

confidence: 99%

Reduced Nuclease Activity of Apurinic/Apyrimidinic Endonuclease (APE1) Variants on Nucleosomes

Hinz

Mao

McNeill

et al. 2015

Background:The apurinic/apyrimidinic endonuclease 1 (APE1) endonuclease of base excision repair must access DNA damage within chromatin. Results: Two natural variants of APE1 have a greater reduction of nuclease activity on nucleosomes. Conclusion: Some amino acid residues in APE1 promote activity in the context of other proteins on DNA. Significance: Knowing how enzymes tolerate protein obstructions on DNA is fundamental to understanding DNA repair in chromatin.