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Abstract Bacteriophage T4 gene 32 protein (gp32) is a model single-stranded DNA (ssDNA) binding protein, essential for DNA replication. gp32 forms cooperative filaments on ssDNA through interprotein interactions between its core and N-terminus. However, detailed understanding of gp32 filament structure and organization remains incomplete, particularly for longer, biologically-relevant DNA lengths. Moreover, it is unclear how these tightly-bound filaments dissociate from ssDNA during complementary strand synthesis. We use optical tweezers and atomic force microscopy to probe the structure and binding dynamics of gp32 on long (∼8 knt) ssDNA substrates. We find that cooperative binding of gp32 rigidifies ssDNA while also reducing its contour length, consistent with the ssDNA helically winding around the gp32 filament. While measured rates of gp32 binding and dissociation indicate nM binding affinity, at ∼1000-fold higher protein concentrations gp32 continues to bind into and restructure the gp32–ssDNA filament, leading to an increase in its helical pitch and elongation of the substrate. Furthermore, the oversaturated gp32–ssDNA filament becomes progressively unwound and unstable as observed by the appearance of a rapid, noncooperative protein dissociation phase not seen at lower complex saturation, suggesting a possible mechanism for prompt removal of gp32 from the overcrowded ssDNA in front of the polymerase during replication.
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The L1-ORF1p coiled coil enables formation of a tightly compacted nucleic acid-bound complex that is associated with retrotransposition
Abstract Long interspersed nuclear element 1 (L1) parasitized most vertebrates and constitutes ∼20% of the human genome. It encodes ORF1p and ORF2p which form an L1-ribonucleoprotein (RNP) with their encoding transcript that is copied into genomic DNA (retrotransposition). ORF1p binds single-stranded nucleic acid (ssNA) and exhibits NA chaperone activity. All vertebrate ORF1ps contain a coiled coil (CC) domain and we previously showed that a CC-retrotransposition null mutant prevented formation of stably bound ORF1p complexes on ssNA. Here, we compared CC variants using our recently improved method that measures ORF1p binding to ssDNA at different forces. Bound proteins decrease ssDNA contour length and at low force, retrotransposition-competent ORF1ps (111p and m14p) exhibit two shortening phases: the first is rapid, coincident with ORF1p binding; the second is slower, consistent with formation of tightly compacted complexes by NA-bound ORF1p. In contrast, two retrotransposition-null CC variants (151p and m15p) did not attain the second tightly compacted state. The C-terminal half of the ORF1p trimer (not the CC) contains the residues that mediate NA-binding. Our demonstrating that the CC governs the ability of NA-bound retrotransposition-competent trimers to form tightly compacted complexes reveals the biochemical phenotype of these coiled coil mutants.
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- Award ID(s):
- 1817712
- PAR ID:
- 10369965
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Nucleic Acids Research
- Volume:
- 50
- Issue:
- 15
- ISSN:
- 0305-1048
- Page Range / eLocation ID:
- p. 8690-8699
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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